Glycopegylated erythropoietin formulations

ABSTRACT

The present invention provides conjugates between erythropoietin and PEG moieties. The conjugates are linked via an intact glycosyl linking group interposed between and covalently attached to the peptide and the modifying group. The conjugates are formed from glycosylated peptides by the action of a glycosyltransferase. The glycosyltransferase ligates a modified sugar moiety onto a glycosyl residue on the peptide. Also provided are methods for preparing the conjugates, methods for treating various disease conditions with the conjugates, and pharmaceutical formulations including the conjugates.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication No. 60/684,637 filed May 25, 2005, U.S. Provisional PatentApplication No. 60/685,007 filed May 25, 2005, U.S. Provisional PatentApplication No. 60/687,548 filed Jun. 2, 2005, U.S. patent applicationSer. No. 11/144,223 filed Jun. 2, 2005, U.S. Provisional PatentApplication No. 60/764,625 filed Feb. 1, 2006, U.S. Provisional PatentApplication No. 60/773,941 filed Feb. 15, 2006 and U.S. ProvisionalPatent Application No. 60/774,088 filed Feb. 15, 2006, each of which isincorporated herein by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Erythropoietin (EPO) is a cytokine produced by the kidney and liverwhich acts on hematopoietic stem cells to stimulate the production ofred blood cells. The protein exists in two forms: one is a 165 aminoacid peptide, and the other is a 166 amino acid peptide. The 166 aminoacid peptide has the same sequence as the 165 amino acid but with anadditional arginine in the most C-terminal position. The mature 165amino acid peptide is a 34 kD glycoprotein comprising threeN-glycosylation sites (Asn-24, Asn-38, and Asn-83), and 1O-glycosylation site (Ser-126). Some variants are “hyperglycosylated”comprising 5 N-linked glycosylation sites.

Erythropoietin synthesis is induced by conditions that effectivelycreate tissue hypoxia, such as lowering of the arterial O₂ tension orincreasing the oxygen affinity of the blood. Under usual conditions ofhomeostasis, hematocrit and the concentration of hemoglobin in blood aremaintained constant with erythropoiesis counterbalancing the permanentdestruction of aged red blood cells by macrophages in bone marrow,spleen and liver. Quantitatively, about 1% of the red cell mass, whichis about 2-3×10¹¹ red blood cells, is renewed each day. However, insituations that effectively generate tissue hypoxia, such as blood lossor location to high altitudes, the induction of EPO may stimulateerythropoiesis 10-fold or more over normal levels.

Because EPO stimulates red blood cell production it is an effectivetherapy for many diseases and conditions associated with reducedhematocrit. Initial trials of replacement therapy with recombinant humanEPO to restore the hematocrit in patients with end-stage renal failurewere first reported about 20 years ago (see e.g., Winearls, C. G.; etal. (1986) Lancet, 2, 1175-1178, and Eschbach, J. W.; et al. (1987) N.Engl. J. Med., 316, 73-78). This work provided an impetus for furtherstudies into the pathophysiology and pharmacology of EPO (see e.g.,Jelkmann, W. and Gross, A. (1989) ERYTHROPOIETIN; Springer, BerlinHeidelberg New York).

Since those early studies, recombinant human EPO has been usedsuccessfully to treat numerous pathological conditions. For example, thepharmacological application of recombinant human EPO to surgicalpatients can lower the severity and duration of postoperative anemia.The administration of recombinant human EPO has also proven to beeffective therapy for patients suffering from several non-renaldiseases, such as chronic inflammation, malignancy and AIDS, wherein arelative lack of endogenous EPO contributes to the development of anemia(see e.g., Means, R. T. and Krantz, S. B. (1992) Blood, 80, 1639-1647,and Jelkmann, W. (1998) J. Interf. Cytokine Res., 18, 555-559).Furthermore, it has been reported that EPO is tissue protective inischemic, traumatic, toxic and inflammatory injuries (see e.g., BrinesM., et al. (2004) Proc. Natl. Acad. Sci. USA 101, 14907-14912 andBrines, M. L., et al. (2000). Proc. Natl. Acad. Sci. USA 97,10526-10531).

The usefulness and effectiveness of EPO for the treatment of anemias andother conditions arising from such a wide variety of causes makesrecombinant human EPO perhaps the best selling drug in the world.Indeed, estimated sales amount to more than 5 billion US dollars peryear.

Recombinant human EPO, produced in Chinese Hamster Ovary (CHO) cellline, is used extensively as a therapeutic. Since mammals all produceglycans of similar structure, Chinese Hamster Ovary (CHO), Baby HamsterKidney (BHK), and Human Embryonic Kidney-293 (HEK-293) are the preferredhost cells for production of glycoprotein therapeutics. As is known inthe art, proper glycosylation is a critically important factorinfluencing the in vivo the half life and immunogenicity of therapeuticpeptides. Poorly glycosylated proteins are recognized by the liver asbeing “old” and thus, are more quickly eliminated from the body than areproperly glycosylated proteins.

Another phenomena that hampers the use of therapeutic peptides is therelatively short in vivo half life exhibited by these peptides. Overall,the problem of short in vivo half life means that therapeuticglycopeptides must be administered frequently in high dosages, whichultimately translate to higher health care costs than might be necessaryif a more efficient method for making longer lasting, more effectiveglycoprotein therapeutics was available.

One solution to the problem of providing cost effective glycopeptidetherapeutics is increasing the in vivo half life of the peptide. Forexample, glycopeptide therapeutics with improved pharmacokineticproperties are produced by attaching synthetic polymers to the peptidebackbone. An exemplary polymer that has been conjugated to peptides ispoly(ethylene glycol) (“PEG”). The use of PEG to derivatize peptidetherapeutics has been demonstrated to reduce the immunogenicity of thepeptides. For example, U.S. Pat. No. 4,179,337 (Davis et al.) disclosesnon-immunogenic polypeptides such as enzymes and peptide hormonescoupled to polyethylene glycol (PEG) or polypropylene glycol. Inaddition to reduced immunogenicity, the clearance time in circulation isprolonged due to the increased size of the PEG-conjugate of thepolypeptides in question.

The principal mode of attachment of PEG, and its derivatives, topeptides is a non-specific covalent bonding through a peptide amino acidresidue (see e.g., U.S. Pat. No. 4,088,538 U.S. Pat. No. 4,496,689, U.S.Pat. No. 4,414,147, U.S. Pat. No. 4,055,635, and PCT WO 87/00056).Another mode of attaching PEG to peptides is through the non-specificoxidation of glycosyl residues on a glycopeptide (see e.g., WO94/05332), which is followed by the reductive amination of the resultingcarbonyl moiety with an amino-PEG species.

In these non-specific methods, poly(ethylene glycol) is added in arandom, non-specific manner to reactive residues on a peptide backbone.Random attachment of PEG molecules has drawbacks, including a lack ofhomogeneity of the final product, and the possibility for reduction inthe biological or enzymatic activity of the peptide. Therefore, for theproduction of therapeutic peptides, a derivitization strategy thatresults in the formation of a specifically labeled, readilycharacterizable, essentially homogeneous PEGylated peptide is superior.As set forth herein, such methods have been developed.

Specifically labeled, homogeneous peptide therapeutics can be producedin vitro through the action of enzymes. Unlike the typical non-specificmethods for attaching a synthetic polymer or other label to a peptide,enzyme-based syntheses have the advantages of regioselectivity andstereoselectivity. Two principal classes of enzymes for use in thesynthesis of labeled peptides are glycosyltransferases (e.g.,sialyltransferases, oligosaccharyltransferases,N-acetylglucosaminyltransferases), and glycosidases. These enzymes canbe used for the specific attachment of sugars which can be subsequentlymodified to comprise a therapeutic moiety. Alternatively,glycosyltransferases and modified glycosidases can be used to directlytransfer modified sugars to a peptide backbone (see e.g., U.S. Pat. No.6,399,336, and U.S. Patent Application Publications 20030040037,20040132640, 20040137557, 20040126838, and 20040142856, each of whichare incorporated by reference herein). Methods combining both chemicaland enzymatic synthetic elements are also known (see e.g., Yamamoto etal. Carbohydr. Res. 305: 415-422 (1998) and U.S. Patent ApplicationPublication 20040137557 which is incorporated herein by reference).

As discussed above, erythropoietin (EPO) is an extremely valuabletherapeutic peptide. Although commercially available forms of EPO are inuse today, these peptides are less than maximally effective due factorsincluding microheterogeneity of the glycoprotein product which increasesproduction costs, poor pharmacokinetics of the resulting isolatedglycoprotein product, or a combination of the two. Thus, there remains aneed in the art for long lasting EPO peptides with improvedeffectiveness and better pharmacokinetics. Furthermore, to be effectivefor the largest number of individuals, it must be possible to produce,on an industrial scale, an EPO peptide with improved therapeuticpharmacokinetics that has a predictable, essentially homogeneous,structure which can be readily reproduced over, and over again.

Fortunately, EPO peptides with improved therapeutic effectiveness andmethods for making them have now been discovered. The present inventionprovides EPO peptides with improved pharmacokinetics. The invention alsoprovides industrially practical and cost effective methods for theproduction of modified EPO peptides. The EPO peptides of the inventioncomprise modifying groups such as PEG moieties, therapeutic moieties,biomolecules and the like. The present invention therefore fulfills theneed for EPO peptides with improved the therapeutic effectiveness andimproved pharmacokinetics for the treatment of conditions and diseaseswherein EPO provides effective therapy.

SUMMARY OF THE INVENTION

It has now been discovered that the controlled modification oferythropoietin (EPO) with one or more polymeric modifying moiety, e.g.,poly(ethylene glycol), affords novel EPO derivatives with improvedpharmacokinetic properties. Furthermore, cost effective methods forreliable and reproducible production of the polymer-modified EPOpeptides of the invention have been discovered and developed.

The polymeric modifying moiety can be attached at any position of aglycosyl moiety of EPO. Moreover, the polymeric modifying moiety can bebound to a glycosyl residue at any position in the amino acid sequenceof a wild type or mutant EPO peptide.

In an exemplary embodiment, the invention provides an EPO peptide thatis conjugated through a glycosyl linking group to a polymeric modifyingmoiety. Exemplary EPO peptide conjugates include a glycosyl linkinggroup having a formula selected from:

In Formulae I and II, R² is H, CH₂OR⁷, COOR⁷ or OR⁷, in which R⁷represents H, substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl. The symbols R³, R⁴, R⁵, R⁶ and R^(6′)independently represent H, substituted or unsubstituted alkyl, OR⁸,NHC(O)R⁹. The index d is 0 or 1. R⁸ and R⁹ are independently selectedfrom H, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl or sialic acid. At least one of R³, R⁴, R⁵, R⁶ or R^(6′)includes the polymeric modifying moiety e.g., PEG. In an exemplaryembodiment, R⁶ and R^(6′), together with the carbon to which they areattached are components of the side chain of sialic acid. In a furtherexemplary embodiment, this side chain is functionalized with thepolymeric modifying moiety.

In an exemplary embodiment, the polymeric moiety is bound to theglycosyl linking group, generally through a heteroatom on the glycosylcore (e.g., N, O), through a linker, L, as shown below:

R¹ is the polymeric modifying moiety and L is selected from a bond and alinking group. The index w represents an integer selected from 1-6,preferably 1-3 and more preferably 1-2. Exemplary linking groups includesubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl moieties and sialic acid. An exemplary component of thelinker is an acyl moiety. Another exemplary linking group is an aminoacid residue (e.g., cysteine, serine, lysine, and short oligopeptides,e.g., Lys-Lys, Lys-Lys-Lys, Cys-Lys, Ser-Lys, etc.)

When L is a bond, it is formed by reaction of a reactive functionalgroup on a precursor of R¹ and a reactive functional group ofcomplementary reactivity on a precursor of the glycosyl linking group.When L is a non-zero order linking group, L can be in place on theglycosyl moiety prior to reaction with the R¹ precursor. Alternatively,the precursors of R¹ and L can be incorporated into a preformed cassettethat is subsequently attached to the glycosyl moiety. As set forthherein, the selection and preparation of precursors with appropriatereactive functional groups is within the ability of those skilled in theart. Moreover, coupling of the precursors proceeds by chemistry that iswell understood in the art.

In an exemplary embodiment L is a linking group that is formed from anamino acid, or small peptide (e.g., 1-4 amino acid residues) providing amodified sugar in which the polymeric modifying moiety is attachedthrough a substituted alkyl linker. Exemplary linkers include glycine,lysine, serine and cysteine. Amino acid analogs, as defined herein, arealso of use as linker components. The amino acid may be modified with anadditional component of a linker, e.g., alkyl, heteroalkyl, covalentlyattached through an acyl linkage, for example, an amide or urethaneformed through an amine moiety of the amino acid residue.

In an exemplary embodiment, the glycosyl linker has a structureaccording to Formula I and R⁵ includes the polymeric modifying moiety.In another exemplary embodiment, R⁵ includes both the polymericmodifying moiety and a linker, L, joining the modifying moiety to theglycosyl core. L can be a linear or branched structure. Similarly, thepolymeric modifying can be branched or linear.

The polymeric modifying moiety comprises two or more repeating unitsthat can be water-soluble or essentially insoluble in water. Exemplarywater-soluble polymers of use in the compounds of the invention includePEG, e.g., m-PEG, PPG, e.g., m-PPG, polysialic acid, polyglutamate,polyaspartate, polylysine, polyethyeleneimine, biodegradable polymers(e.g., polylactide, polyglyceride), and functionalized PEG, e.g.,terminal-functionalized PEG.

The glycosyl core of the glycosyl linking groups of use in the EPOconjugates of the invention is selected from both natural and unnaturalfuranoses and pyranoses. The unnatural saccharides optionally include analkylated or acylated hydroxyl and/or amine moiety, e.g., ethers, estersand amide substituents on the ring. Other unnatural saccharides includean H, hydroxyl, ether, ester or amide substituent at a position on thering at which such a substituent is not present in the naturalsaccharide. Alternatively, the carbohydrate is missing a substituentthat would be found in the carbohydrate from which its name is derived,e.g., deoxy sugars. Still further exemplary unnatural sugars includeboth oxidized (e.g., -onic and -uronic acids) and reduced (sugaralcohols) carbohydrates. The sugar moiety can be a mono-, oligo- orpoly-saccharide.

Exemplary natural sugars of use as components of glycosyl linking groupsin the present invention include glucose, glucosamine, galactose,galactosamine, fucose, mannose, mannosamine, xylanose, ribose, N-acetylglucose, N-acetyl glucosamine, N-acetyl galactose, N-acetylgalactosamine, and sialic acid.

In one embodiment, the present invention provides an erythropoietinpeptide comprising the moiety:

wherein D is a member selected from —OH and R¹-L-HN—; G is a memberselected from H and R′-L- and —C(O)(C₁-C₆)alkyl; R¹ is a moietycomprising a straight-chain or branched poly(ethylene glycol) residue;and L is a linker, e.g., a bond (“zero order”), substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl. Inexemplary embodiments, when D is OH, G is R¹-L-, and when G is—C(O)(C₁-C₆)alkyl, D is R¹-L-NH—.

In another aspect, the invention provides a peptide comprising aglycosyl linking group having the formula:

In other embodiments, the group has the formula:

in which t is 0 or 1.

In yet another embodiment, the group has the formula:

in which the index p represents and integer from 1 to 10, and arepresents 0 or 1.

In another aspect, the invention provides a method of making a PEGylatederythropoietin of the invention. The method includes: (a) contacting asubstrate erythropoietin peptide comprising a glycosyl group selectedfrom:

with a PEG-sialic acid donor having the formula:

and an enzyme that transfers PEG-sialic acid from said donor onto amember selected from the Gal and the Sia of said glycosyl group, underconditions appropriate for said transfer. An exemplary modified sialicacid donor is CMP-sialic acid modified, through a linker moiety, with apolymer, e.g., a straight chain or branched poly(ethylene glycol)moiety.

The peptide can be acquired from essentially any source, however, in oneembodiment, prior to being modified as discussed above, theerythropoietin peptide is expressed in a suitable host. Mammalian (e.g.,CHO) and insect cells (e.g., Sf-9) are exemplary expression systemsproviding EPO of use in the compositions and methods set forth herein.

In another aspect, the invention provides a method of treating acondition in a subject in need thereof. Exemplary conditions includethose characterized by compromised red blood cell production in thesubject. The method includes the step of administering to the subject anamount of the polymer-modified erythropoietin peptide of the inventioneffective to ameliorate the condition in the subject.

In another aspect, the invention provides a method of enhancing redblood cell production in a mammal. The method includes administering tothe mammal an amount of the polymer-modified erythropoietin peptide ofthe invention effective to enhance red blood cell production in themammal.

In another aspect, the invention provides a method of treating a tissueinjury in a subject in need thereof. Exemplary injuries include thosecharacterized by damage resulting from ischemia, trauma, inflammation orcontact with toxic substances. The method includes the step ofadministering to the subject an amount of a polymer-modifiederythropoietin peptide of the invention effective to ameliorate thetissue injury in the subject. An exemplary class of protection ortreatment includes neuroprotection (e.g., treatment of stroke,Alzheimer's, Parkinson's and other degenerative neurological disorders).The modified EPO of the invention is also of use in treating patientswith diseases such as compromised kidney function, cancer, andretinopathy.

In another aspect, the invention provides a pharmaceutical formulationcomprising a polymer-modified erythropoietin peptide of the inventionand the formulation is substantially stable for storage, handling,and/or therapeutic applications.

In another aspect, the invention provides a pharmaceutical formulationcomprising a polymer-modified erythropoietin peptide of the inventionand a pharmaceutically acceptable carrier.

In another aspect, the invention provides an erythropoietin formulationcomprising a polymer-modified erythropoietin peptide of the inventionand a buffer, e.g., to provide a suitable pH range for the formulation.

In another aspect, the invention provides an erythropoietin formulationcomprising a polymer-modified erythropoietin peptide of the inventionand a tonicity adjusting agent

In another aspect, the invention provides an erythropoietin formulationcomprising a polymer-modified erythropoietin peptide of the inventionand a surfactant.

In another aspect, the invention provides an erythropoietin formulationcomprising a polymer-modified erythropoietin peptide of the inventionand at least one stabilizer, including, but not limited to, generalstabilizers that stabilize by preferential hydration, and specificstabilizers such as metal chelating agents, antioxidants orantimicrobial preservatives.

In the polymer-modified erythropoietin glycoconjugates of the invention,essentially each of the amino acid residues to which the polymer isbound has the same structure across the population individual peptidemolecules. For example, if one peptide molecule includes a Ser linkedglycosyl residue that includes a glycosyl linking group attached to apolymeric modifying moiety, at least about 70%, 80%, 90%, 95%, 97%, 99%,99.2%, 99.4%, 99.6%, or more preferably 99.8% of the other peptides inthe population will have the same glycosyl residue with the polymericmodifying moiety covalently bound to the same Ser residue.

Other objects and advantages of the invention will be apparent to thoseof skill in the art from the detailed description that follows.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates exemplary modified sialic acid nucleotides useful inthe practice of the invention. A. Structure of exemplary branched (e.g.,30 kDa, 40 kDa) CMP-sialic acid-PEG sugar nucleotides. B. Structure oflinear CMP-sialic acid-PEG (e.g., 10 kDa).

FIG. 2 is a representation of exemplary glycoPEGylated EPO isoformsisolated from Chinese Hamster Ovary cells. A. An exemplary O- orN-linked PEGylated glycoform. B. Is a representation of exemplary EPOisoforms isolated from insect cells and remodeled and glycoPEGylated.FIG. 2A and FIG. 2B are exemplary in that any glycosylated EPO moleculemay comprise any mixture of mono-, bi- tri-, or tetra-antennary N-linkedglycosyl residues and any one or more of the branches may furthercomprise a modified sialic acid moiety. Moreover, the modified glycancan be positioned at any one or more N- or O-linked glycosylation sitewithout limitation. Each of the indices is independently selected from 0and 1, and R¹ is as described herein. The peptide includes at least oneR¹⁵ moiety that includes a branched or linear PEG moiety. In this andeach of the other figures in which the symbol

appears, it represents a discontinuity in the representation of thepeptide chain due to the size of the drawing. The representation iscontinued on the subsequent line. The symbol does not imply an actualbreak in the peptide sequence.

FIG. 3 illustrates an exemplary CHO-derived EPO peptide in itsnon-glycoPEGylated form. The figure is exemplary in that anyglycosylated EPO molecule may comprise any mixture of mono-, bi- tri-,or tetra-antennary N-linked glycosyl residues and any one or more of thebranches may further comprise a modified sialic acid moiety of theinvention. Moreover, the figure illustrates that the modified glycan canbe positioned at any one or more N- or O-linked glycosylation sitewithout limitation.

FIG. 4 shows the results of experiments comparing the pharmacokineticsof two CHO-derived non-glycoPEGylated EPO forms, and two differentCHO-derived glycoPEGylated EPO forms.

FIG. 5 illustrates an insect-derived remodeled and glycoPEGylated EPOpeptide according to the invention.

FIG. 6 shows the results of experiments comparing the pharmacokineticsof a CHO-derived non-glycoPEGylated EPO form, an insect-derivednon-glycoPEGylated EPO form, with their corresponding glycoPEGylatedforms.

FIG. 7 shows the relative activites of two forms of non-glycoPEGylatedEPO (A and B) versus two glycoPEGylated variants (the 30 kilodalton and40 kilodalton variants of FIGS. 2A and 2B) and a hyperglycosylated EPOvariant in stimulating proliferation of EPO receptor-bearing TF1 cellsin culture.

FIG. 8 shows inhibition of binding of isotope-labeled EPO to arecombinant chimeric EPO receptor by various concentrations of twonon-pegylated EPO variants (A and B) and two glycoPEGylated variants(the 30 kilodalton and 40 kilodalton variants of FIGS. 2A and 2B).

FIG. 9 is a table displaying sialyltransferases of use to glycoPEGylatepeptides with a modified sialic acid.

FIG. 10 is a diagram outlining the production process for CMP-SA-PEG-30kDa in an exemplary embodiment of the invention. After reaction of theactivated PEG reagent (mPEG-30 kDa-nitrophenyl carbonate) with themodified EPO peptide (CMP-SA-Glycine), the reaction product is desaltedand conditioned for subsequent purification steps by ultrafiltrationemploying a tangential flow filtration (TFF) cascade. In a second stepthe peptide is purified by ion exchange chromatography using aQ-Sepharose resin followed by additional ultrafiltration steps resultingin a desalted EPO-peptide solution. In a final step the product islyophilized to afford CMP-SA-PEG-30 kDa.

DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTS

Abbreviations

PEG, poly(ethylene glycol); PPG, poly(propylene glycol); Ara,arabinosyl; Fru, fructosyl; Fuc, fucosyl; Gal, galactosyl; GalNAc,N-acetylgalactosaminyl; Glc, glucosyl; GlcNAc, N-acetylglucosaminyl;Man, mannosyl; ManAc, mannosaminyl acetate; Xyl, xylosyl; NeuAc(N-acetylneuraminyl), Sia (sialyl); M6P, mannose-6-phosphate.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization are those well known and commonly employedin the art. Standard techniques are used for nucleic acid and peptidesynthesis. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, andorganic synthetic described below are those well known and commonlyemployed in the art. Standard techniques, or modifications thereof, areused for chemical syntheses and chemical analyses.

All oligosaccharides described herein are described with the name orabbreviation for the non-reducing saccharide (i.e., Gal), followed bythe configuration of the glycosidic bond (α or β), the ring bond (1 or2), the ring position of the reducing saccharide involved in the bond(2, 3, 4, 6 or 8), and then the name or abbreviation of the reducingsaccharide (i.e., GlcNAc). Each saccharide is preferably a pyranose. Fora review of standard glycobiology nomenclature, see, Essentials ofGlycobiology Varki et al. eds. CSHL Press (1999).

Oligosaccharides are considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. In accordance with accepted nomenclature,oligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right.

The term “sialic acid” refers to any member of a family of nine-carboncarboxylated sugars. The most common member of the sialic acid family isN-acetyl-neuraminic acid(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member ofthe family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which theN-acetyl group of NeuAc is hydroxylated. A third sialic acid familymember is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J.Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265:21811-21819 (1990)). Also included are 9-substituted sialic acids suchas a 9-O—C₁-C₆ acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki, Glycobiology 2: 25-40 (1992);Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed.(Springer-Verlag, New York (1992)). The synthesis and use of sialic acidcompounds in a sialylation procedure is disclosed in internationalapplication WO 92/16640, published Oct. 1, 1992.

“Peptide” refers to a polymer in which the monomers are amino acids andare joined together through amide bonds, alternatively referred to as apolypeptide. Additionally, unnatural amino acids, for example,β-alanine, phenylglycine and homoarginine are also included. Amino acidsthat are not gene-encoded may also be used in the present invention.Furthermore, amino acids that have been modified to include reactivegroups, glycosylation sites, polymers, therapeutic moieties,biomolecules and the like may also be used in the invention. All of theamino acids used in the present invention may be either the D- orL-isomer. The L-isomer is generally preferred. In addition, otherpeptidomimetics are also useful in the present invention. As usedherein, “peptide” refers to both glycosylated and unglycosylatedpeptides. Also included are peptides that are incompletely glycosylatedby a system that expresses the peptide. For a general review, see,Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDESAND PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267(1983).

The term “peptide conjugate,” refers to species of the invention inwhich a peptide is conjugated with a modified sugar as set forth herein.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amanner similar to a naturally occurring amino acid.

As used herein, the term “modified sugar,” refers to a naturally- ornon-naturally-occurring carbohydrate that is enzymatically added onto anamino acid or a glycosyl residue of a peptide in a process of theinvention. The modified sugar is selected from enzyme substratesincluding, but not limited to sugar nucleotides (mono-, di-, andtri-phosphates), activated sugars (e.g., glycosyl halides, glycosylmesylates) and sugars that are neither activated nor nucleotides. The“modified sugar” is covalently functionalized with a “modifying group.”Useful modifying groups include, but are not limited to, PEG moieties,therapeutic moieties, diagnostic moieties, biomolecules and the like.The modifying group is preferably not a naturally occurring, or anunmodified carbohydrate. The locus of functionalization with themodifying group is selected such that it does not prevent the “modifiedsugar” from being added enzymatically to a peptide.

The term “water-soluble” refers to moieties that have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like. Peptides can have mixed sequencesof be composed of a single amino acid, e.g., poly(lysine). An exemplarypolysaccharide is poly(sialic acid). An exemplary poly(ether) ispoly(ethylene glycol). Poly(ethylene imine) is an exemplary polyamine,and poly(acrylic) acid is a representative poly(carboxylic acid).

The polymer backbone of the water-soluble polymer can be poly(ethyleneglycol) (i.e. PEG). However, it should be understood that other relatedpolymers are also suitable for use in the practice of this invention andthat the use of the term PEG or poly(ethylene glycol) is intended to beinclusive and not exclusive in this respect. The term PEG includespoly(ethylene glycol) in any of its forms, including alkoxy PEG,difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG(i.e. PEG or related polymers having one or more functional groupspendent to the polymer backbone), or PEG with degradable linkagestherein.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(-PEG-OH)_(m) in which R represents the core moiety,such as glycerol or pentaerythritol, and m represents the number ofarms. Multi-armed PEG molecules, such as those described in U.S. Pat.No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the polymer backbone.

Many other polymers are also suitable for the invention. Polymerbackbones that are non-peptidic and water-soluble, with from 2 to about300 termini, are particularly useful in the invention. Examples ofsuitable polymers include, but are not limited to, other poly(alkyleneglycols), such as poly(propylene glycol) (“PPG”), copolymers of ethyleneglycol and propylene glycol and the like, poly(oxyethylated polyol),poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxypropylmethacrylamide), poly(α-hydroxy acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine),such as described in U.S. Pat. No. 5,629,384, which is incorporated byreference herein in its entirety, and copolymers, terpolymers, andmixtures thereof. Although the molecular weight of each chain of thepolymer backbone can vary, it is typically in the range of from about100 Da to about 100,000 Da, often from about 6,000 Da to about 80,000Da.

The “area under the curve” or “AUC”, as used herein in the context ofadministering a peptide drug to a patient, is defined as total areaunder the curve that describes the concentration of drug in systemiccirculation in the patient as a function of time from zero to infinity.

The term “half-life” or “t½”, as used herein in the context ofadministering a peptide drug to a patient, is defined as the timerequired for plasma concentration of a drug in a patient to be reducedby one half. There may be more than one half-life associated with thepeptide drug depending on multiple clearance mechanisms, redistribution,and other mechanisms well known in the art. Usually, alpha and betahalf-lives are defined such that the alpha phase is associated withredistribution, and the beta phase is associated with clearance.However, with protein drugs that are, for the most part, confined to thebloodstream, there can be at least two clearance half-lives. For someglycosylated peptides, rapid beta phase clearance may be mediated viareceptors on macrophages, or endothelial cells that recognize terminalgalactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, orfucose. Slower beta phase clearance may occur via renal glomerularfiltration for molecules with an effective radius <2 nm (approximately68 kDa) and/or specific or non-specific uptake and metabolism intissues. GlycoPEGylation may cap terminal sugars (e.g., galactose orN-acetylgalactosamine) and thereby block rapid alpha phase clearance viareceptors that recognize these sugars. It may also confer a largereffective radius and thereby decrease the volume of distribution andtissue uptake, thereby prolonging the late beta phase. Thus, the preciseimpact of glycoPEGylation on alpha phase and beta phase half-lives mayvary depending upon the size, state of glycosylation, and otherparameters, as is well known in the art. Further explanation of“half-life” is found in Pharmaceutical Biotechnology (1997, DFACrommelin and RD Sindelar, eds., Harwood Publishers, Amsterdam, pp101-120).

The term “glycoconjugation,” as used herein, refers to the enzymaticallymediated conjugation of a modified sugar species to an amino acid orglycosyl residue of a polypeptide, e.g., an Erythropoietin peptide ofthe present invention. A subgenus of “glycoconjugation” is“glyco-PEGylation,” in which the modifying group of the modified sugaris poly(ethylene glycol), and alkyl derivative (e.g., m-PEG) or reactivederivative (e.g., H₂N-PEG, HOOC-PEG) thereof.

The terms “large-scale” and “industrial-scale” are used interchangeablyand refer to a reaction cycle that produces at least about 250 mg,preferably at least about 500 mg, and more preferably at least about 1gram of glycoconjugate at the completion of a single reaction cycle.

The term, “glycosyl linking group,” as used herein refers to a glycosylresidue to which a modifying group (e.g., PEG moiety, therapeuticmoiety, biomolecule) is covalently attached; the glycosyl linking groupjoins the modifying group to the remainder of the conjugate. In themethods of the invention, the “glycosyl linking group” becomescovalently attached to a glycosylated or unglycosylated peptide, therebylinking the agent to an amino acid and/or glycosyl residue on thepeptide. A “glycosyl linking group” is generally derived from a“modified sugar” by the enzymatic attachment of the “modified sugar” toan amino acid and/or glycosyl residue of the peptide. The glycosyllinking group can be a saccharide-derived structure that is degradedduring formation of modifying group-modified sugar cassette (e.g.,oxidation→Schiff base formation→reduction), or the glycosyl linkinggroup may be intact. An “intact glycosyl linking group” refers to alinking group that is derived from a glycosyl moiety in which thesaccharide monomer that links the modifying group and to the remainderof the conjugate is not degraded, e.g., oxidized, e.g., by sodiummetaperiodate. “Intact glycosyl linking groups” of the invention may bederived from a naturally occurring oligosaccharide by addition ofglycosyl unit(s) or removal of one or more glycosyl unit from a parentsaccharide structure.

The term “targeting moiety,” as used herein, refers to species that willselectively localize in a particular tissue or region of the body. Thelocalization is mediated by specific recognition of moleculardeterminants, molecular size of the targeting agent or conjugate, ionicinteractions, hydrophobic interactions and the like. Other mechanisms oftargeting an agent to a particular tissue or region are known to thoseof skill in the art. Exemplary targeting moieties include antibodies,antibody fragments, transferrin, HS-glycoprotein, coagulation factors,serum proteins, O-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO and the like.

As used herein, “therapeutic moiety” means any agent useful for therapyincluding, but not limited to, antibiotics, anti-inflammatory agents,anti-tumor drugs, cytotoxins, and radioactive agents. “Therapeuticmoiety” includes prodrugs of bioactive agents, constructs in which morethan one therapeutic moiety is bound to a carrier, e.g, multivalentagents. Therapeutic moiety also includes proteins and constructs thatinclude proteins. Exemplary proteins include, but are not limited to,Granulocyte Colony Stimulating Factor (GCSF), Granulocyte MacrophageColony Stimulating Factor (GMCSF), Interferon (e.g., Interferon-α, -β,-γ), Interleukin (e.g., Interleukin II), serum proteins (e.g., FactorsVII, VIIa, VIII, IX, and X), Human Chorionic Gonadotropin (HCG),Follicle Stimulating Hormone (FSH) and Lutenizing Hormone (LH) andantibody fusion proteins (e.g. Tumor Necrosis Factor Receptor ((TNFR)/Fcdomain fusion protein)).

As used herein, “pharmaceutically acceptable carrier” includes anymaterial, which when combined with the conjugate retains the conjugates'activity and is non-reactive with the subject's immune systems. Examplesinclude, but are not limited to, any of the standard pharmaceuticalcarriers such as a phosphate buffered saline solution, water, emulsionssuch as oil/water emulsion, and various types of wetting agents. Othercarriers may also include sterile solutions, tablets including coatedtablets and capsules. Typically such carriers contain excipients such asstarch, milk, sugar, certain types of clay, gelatin, stearic acid orsalts thereof, magnesium or calcium stearate, talc, vegetable fats oroils, gums, glycols, or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients. Compositionscomprising such carriers are formulated by well known conventionalmethods.

As used herein, “administering,” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, intranasal orsubcutaneous administration, or the implantation of a slow-releasedevice e.g., a mini-osmotic pump, to the subject. Administration is byany route including parenteral, and transmucosal (e.g., oral, nasal,vaginal, rectal, or transdermal). Parenteral administration includes,e.g., intravenous, intramuscular, intra-arteriole, intradermal,subcutaneous, intraperitoneal, intraventricular, and intracranial.Moreover, where injection is to treat a tumor, e.g., induce apoptosis,administration may be directly to the tumor and/or into tissuessurrounding the tumor. Other modes of delivery include, but are notlimited to, the use of liposomal formulations, intravenous infusion,transdermal patches, etc.

The term “ameliorating” or “ameliorate” refers to any indicia of successin the treatment of a pathology or condition, including any objective orsubjective parameter such as abatement, remission or diminishing ofsymptoms or an improvement in a patient's physical or mental well-being.Amelioration of symptoms can be based on objective or subjectiveparameters; including the results of a physical examination and/or apsychiatric evaluation.

The term “therapy” refers to “treating” or “treatment” of a disease orcondition including preventing the disease or condition from occurringin an animal that may be predisposed to the disease but does not yetexperience or exhibit symptoms of the disease (prophylactic treatment),inhibiting the disease (slowing or arresting its development), providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment), and relieving the disease (causing regression ofthe disease).

The term “effective amount” or “an amount effective to” or a“therapeutically effective amount” or any grammatically equivalent termmeans the amount that, when administered to an animal for treating adisease, is sufficient to effect treatment for that disease.

The term “tissue protective” refers to the defense of a tissue againstthe effects of cellular damage that are typically associated with theexperience by a tissue or organ of ischemia/hypoxia, trauma, toxicityand/or inflammation. Cellular damage may lead to apoptosis and/ornecrosis (i.e., toxic cell death). Thus, a “tissue protective” effectguards a tissue from experiencing the degree of apoptosis and/or toxiccell death normally associated with a given traumatic, inflammatory,toxic or ischemic injury. For example, EPO reduces the area of infarctafter middle cerebral artery occlusion in a rodent model (Siren, A. L.et al. (2001). Proc. Natl. Acad. Sci. U.S.A. 98, 4044-4049). Thus, undersuch conditions EPO provides a “tissue protective” effect by effectivelyreducing the necrosis and/or apoptosis normally associated with theischemic injury (e.g., ischemic stroke). “Tissue protective” also refersto the defense of a tissue against the effects of cellular damage andthe ensuing cell death associated with degenerative diseases such asretinopathy, or neurodegenerative disease.

The term “isolated” refers to a material that is substantially oressentially free from components, which are used to produce thematerial. For peptide conjugates of the invention, the term “isolated”refers to material that is substantially or essentially free fromcomponents which normally accompany the material in the mixture used toprepare the (LC-MS), matrix assisted laser desorption mass time offlight spectrometry (MALDITOF), capillary electrophoresis, and the like.

“Substantially uniform glycoform” or a “substantially uniformglycosylation pattern,” when referring to a glycopeptide species, refersto the percentage of acceptor moieties that are glycosylated by theglycosyltransferase of interest (e.g., fucosyltransferase). For example,in the case of a α1,2 fucosyltransferase, a substantially uniformfucosylation pattern exists if substantially all (as defined below) ofthe Galβ1,4-GlcNAc-R and sialylated analogues thereof are fucosylated ina peptide conjugate of the invention. In the fucosylated structures setforth herein, the Fuc-GlcNAc linkage is generally α1,6 or α1,3, withα1,6 generally preferred. It will be understood by one of skill in theart, that the starting material may contain glycosylated acceptormoieties (e.g., fucosylated Galβ1,4-GlcNAc-R moieties). Thus, thecalculated percent glycosylation will include acceptor moieties that areglycosylated by the methods of the invention, as well as those acceptormoieties already glycosylated in the starting material.

The term “substantially” in the above definitions of “substantiallyuniform” generally means at least about 40%, at least about 70%, atleast about 80%, or more preferably at least about 90%, and still morepreferably at least about 95% of the acceptor moieties for a particularglycosyltransferase are glycosylated.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-peptide conjugate. “Isolated” and “pure” are usedinterchangeably. Typically, isolated peptide conjugates of the inventionhave a level of purity preferably expressed as a range. The lower end ofthe range of purity for the peptide conjugates is about 60%, about 70%or about 80% and the upper end of the range of purity is about 70%,about 80%, about 90% or more than about 90%.

When the peptide conjugates are more than about 90% pure, their puritiesare also preferably expressed as a range. The lower end of the range ofpurity is about 90%, about 92%, about 94%, about 96% or about 98%. Theupper end of the range of purity is about 92%, about 94%, about 96%,about 98% or about 100% purity.

Purity is determined by any art-recognized method of analysis (e.g.,band intensity on a silver stained gel, polyacrylamide gelelectrophoresis, HPLC, or a similar means).

“Essentially each member of the population,” as used herein, describes acharacteristic of a population of peptide conjugates of the invention inwhich a selected percentage of the modified sugars added to a peptideare added to multiple, identical acceptor sites on the peptide.“Essentially each member of the population” speaks to the “homogeneity”of the sites on the peptide conjugated to a modified sugar and refers toconjugates of the invention, which are at least about 80%, preferably atleast about 90% and more preferably at least about 95% homogenous.

“Homogeneity,” refers to the structural consistency across a populationof acceptor moieties to which the modified sugars are conjugated. Thus,in a peptide conjugate of the invention in which each modified sugarmoiety is conjugated to an acceptor site having the same structure asthe acceptor site to which every other modified sugar is conjugated, thepeptide conjugate is said to be about 100% homogeneous. Homogeneity istypically expressed as a range. The lower end of the range ofhomogeneity for the peptide conjugates is about 60%, about 70% or about80% and the upper end of the range of purity is about 70%, about 80%,about 90% or more than about 90%.

When the peptide conjugates are more than or equal to about 90%homogeneous, their homogeneity is also preferably expressed as a range.The lower end of the range of homogeneity is about 90%, about 92%, about94%, about 96% or about 98%. The upper end of the range of purity isabout 92%, about 94%, about 96%, about 98% or about 100% homogeneity.The purity of the peptide conjugates is typically determined by one ormore methods known to those of skill in the art, e.g., liquidchromatography-mass spectrometry (1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃; —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, substituent that can be a single ring or multiple rings(preferably from 1 to 3 rings), which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) is meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″ R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—CR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂in a number ranging from zero to (2m′+1), where m′ is the total numberof carbon atoms in such radical. R′, R″, R′″ and R″″ each preferablyindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″ R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(u)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and u is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(z)—X—(CR″R′″)_(d)—, where z and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

Introduction

Erythropoietin (EPO) is a glycoprotein which serves as the principalregulator of red blood cell synthesis. Erythropoietin acts bystimulating precursor cells in the bone marrow causing them to divideand differentiate into mature red blood cells. EPO may exist as either a165 or 166 amino acid glycoprotein. The 166 amino acid variant isdistinguished from the 165 amino acid variant by the presence of anadditional arginine residue at the C-terminal end of the protein.

Recombinant EPO has been available for some time and is an effectivetherapeutic agent in the treatment of various forms of anemia, includinganemias associated with chronic renal failure, zidovidine treated HIVinfected patients, and cancer patients on chemotherapy. The glycoproteinis administered parenterally, either as an intravenous (IV) orsubcutaneous (SC) injection.

To improve the effectiveness of recombinant erythropoietin used fortherapeutic purposes, the present invention provides polymer conjugatesof glycosylated and unglycosylated erythropoietin peptides. Theconjugates may be additionally modified by further conjugation withdiverse species such as therapeutic moieties, diagnostic moieties,targeting moieties and the like.

The conjugates of the invention are formed by the enzymatic attachmentof a modified sugar bearing the polymeric modifying moiety to theglycosylated or unglycosylated peptide. Glycosylation sites provide locifor conjugating polymeric and other modifying groups to the peptide,e.g., by glycoconjugation. An exemplary modifying group is awater-soluble polymer, such as poly(ethylene glycol), e.g.,methoxy-poly(ethylene glycol). Modification of the EPO peptides canimprove the stability and retention time of the recombinant EPO in apatient's circulation and/or reduce the antigenicity of recombinant EPO.

The invention provides EPO peptides and glycopeptides that have asubstantially homogeneous derivatization pattern. The invention alsoprovides methods of preparing such peptides. The enzymes used in themethods of the invention are generally selective for a particular aminoacid residue, combination of amino acid residues, or particular glycosylresidues of the peptide. The methods are also practical for large-scaleproduction of modified peptides and glycopeptides. Thus, the methods ofthe invention provide a practical means for large-scale preparation ofglycopeptides having preselected uniform derivatization patterns.

The present invention also provides conjugates of glycosylated andunglycosylated peptides with increased therapeutic half-life due to, forexample, reduced clearance rate, or reduced rate of uptake by the immuneor reticuloendothelial system (RES). Moreover, the methods of theinvention provide a means for masking antigenic determinants onpeptides, thus reducing or eliminating a host immune response againstthe peptide. Selective attachment of targeting agents can also be usedto target a peptide to a particular tissue or cell surface receptor thatis specific for the particular targeting agent.

The Conjugates

In a first aspect, the present invention provides a conjugate between aselected modifying group and an EPO peptide. The link between thepeptide and the modifying moiety includes a glycosyl linking groupinterposed between the peptide and the selected moiety. As discussedherein, the selected modifying moiety is essentially any species thatcan be attached to a saccharide unit, resulting in a “modified sugar”that is recognized by an appropriate transferase enzyme, which appendsthe modified sugar onto the peptide, or a glycosyl residue attachedthereto. The saccharide component of the modified sugar, when interposedbetween the peptide and a selected moiety, becomes a “glycosyl linkinggroup,” e.g., an “intact glycosyl linking group.” The glycosyl linkinggroup is formed from any mono- or oligo-saccharide that, aftermodification with the modifying group, is a substrate for an enzyme thatadds the modified sugar to an amino acid or glycosyl residue of apeptide.

The glycosyl linking group can be, or can include, a saccharide moietythat is degradatively modified before or during the addition of themodifying group. For example, the glycosyl linking group can be derivedfrom a saccharide residue that is produced by oxidative degradation ofan intact saccharide to the corresponding aldehyde, e.g., via the actionof metaperiodate, and subsequently converted to a Schiff base with anappropriate amine, which is then reduced to the corresponding amine.

The conjugates of the invention will typically correspond to the generalstructure:

in which the symbols a, b, c, d and s represent a positive, non-zerointeger; and t is either 0 or a positive integer. The “agent” is atherapeutic agent, a bioactive agent, a detectable label, water-solublemoiety (e.g., PEG, m-PEG, PPG, and m-PPG) or the like. The “agent” canbe a peptide, e.g., enzyme, antibody, antigen, etc. The linker can beany of a wide array of linking groups, infra. Alternatively, the linkermay be a single bond or a “zero order linker.”

In an exemplary embodiment, the selected modifying group is awater-soluble polymer, e.g., m-PEG. The water-soluble polymer iscovalently attached to the peptide via a glycosyl linking group. Theglycosyl linking group is covalently attached to an amino acid residueor a glycosyl residue of the peptide. The invention also providesconjugates in which an amino acid residue and a glycosyl residue aremodified with a glycosyl linking group.

An exemplary water-soluble polymer is poly(ethylene glycol), e.g.,methoxy-poly(ethylene glycol). The poly(ethylene glycol) used in thepresent invention is not restricted to any particular form or molecularweight range. For unbranched poly(ethylene glycol) molecules themolecular weight is preferably between 500 and 100,000. A molecularweight of 2000-60,000 is preferably used and preferably of from about5,000 to about 30,000.

In another embodiment the poly(ethylene glycol) is a branched PEG havingmore than one PEG moiety attached. Examples of branched PEGs aredescribed in U.S. Pat. No. 5,932,462; U.S. Pat. No. 5,342,940; U.S. Pat.No. 5,643,575; U.S. Pat. No. 5,919,455; U.S. Pat. No. 6,113,906; U.S.Pat. No. 5,183,660; WO 02/09766; Kodera Y., Bioconjugate Chemistry 5:283-288 (1994); and Yamasaki et al., Agric. Biol. Chem., 52: 2125-2127,1998. In a preferred embodiment the molecular weight of eachpoly(ethylene glycol) of the branched PEG is less than or equal to40,000 daltons.

In addition to providing conjugates that are formed through anenzymatically added glycosyl linking group, the present inventionprovides conjugates that are highly homogenous in their substitutionpatterns. Using the methods of the invention, it is possible to formpeptide conjugates in which essentially all of the modified sugarmoieties across a population of conjugates of the invention are attachedto a structurally identical amino acid or glycosyl residue. Thus, in asecond aspect, the invention provides a peptide conjugate having apopulation of water-soluble polymer moieties, which are covalently boundto the peptide through a glycosyl linking group, e.g., an intactglycosyl linking group. In a preferred conjugate of the invention,essentially each member of the population is bound via the glycosyllinking group to a glycosyl residue of the peptide, and each glycosylresidue of the peptide to which the glycosyl linking group is attachedhas the same structure.

Also provided is a peptide conjugate having a population ofwater-soluble polymer moieties covalently bound thereto through aglycosyl linking group. In a preferred embodiment, essentially everymember of the population of water soluble polymer moieties is bound toan amino acid residue of the peptide via a glycosyl linking group, andeach amino acid residue having a glycosyl linking group attached theretohas the same structure.

The present invention also provides conjugates analogous to thosedescribed above in which the peptide is conjugated to a therapeuticmoiety, diagnostic moiety, targeting moiety, toxin moiety or the likevia an intact glycosyl linking group. Each of the above-recited moietiescan be a small molecule, natural polymer (e.g., polypeptide) orsynthetic polymer. When the modifying moiety is attached to a sialicacid, it is generally preferred that the modifying moiety issubstantially non-fluorescent.

Essentially any erythropoietin peptide having any sequence is of use asa component of the conjugates of the present invention. In an exemplaryembodiment, the peptide has the sequence: (SEQ ID NO:1) H₂N-APPRLICDSRVLERYLLEAK EAENITTGCA EHCSLNENIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALLSEAVLRGQAL LVNSSQPWEP LQLHVDKAVS GLRSLTTLLR ALGAQKEAIS PPDAASAAPLRTITADTFRK LFRVYSNFLR GKLGKYTGEA CRTGD-COOH.

In another exemplary embodiment the peptide has the sequence: (SEQ IDNO:2) H₂N-APPRLICDSR VLERYLLEAK EAENITTGCA EHCSLNENIT VPDTKVNFYAWKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVNSSQPWEP LQLHVDKAVS GLRSLTTLLRALGAQKEAIS PPDAASAAPL RITTADTFRK LFRVYSNFLR GKLKLYTGEA CRTGD-COOH.

Preferably, neither the amino nor the carboxy terminus of the EPOpeptide is derivatized with a polymeric modifying moiety.

The peptides of the invention include at least one N-linked or O-linkedglycosylation site, which is glycosylated with a glycosyl residue thatincludes a polymeric modifying moiety, e.g., a PEG moiety. In anexemplary embodiment, the PEG is covalently attached to the peptide viaan intact glycosyl linking group. The glycosyl linking group iscovalently attached to either an amino acid residue or a glycosylresidue of the peptide. Alternatively, the glycosyl linking group isattached to one or more glycosyl units of a glycopeptide. The inventionalso provides conjugates in which a glycosyl linking group is attachedto both an amino acid residue and a glycosyl residue.

The PEG moiety is attached to an intact glycosyl linker directly, or viaa non-glycosyl linker, e.g., substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl.

In an exemplary embodiment, the invention utilizes a glycosyl linkinggroup that has the formula:

in which J is a glycosyl moiety, L is a bond or a linker and R¹ is amodifying group, e.g., a polymeric modifying group. Exemplary bonds arethose that are formed between an NH₂ moiety on the glycosyl moiety and agroup of complementary reactivity on the modifying group. For example,when R¹ includes a carboxylic acid moiety, this moiety may be activatedand coupled with the NH₂ moiety on the glycosyl residue affording a bondhaving the structure NHC(O)R¹. J is preferably a glycosyl moiety that is“intact”, not having been degraded by exposure to conditions that cleavethe pyranose or furanose structure, e.g. oxidative conditions, e.g.,sodium periodate.

Exemplary linkers include alkyl and heteroalkyl moieties. The linkersinclude linking groups, for example acyl-based linking groups, e.g.,—C(O)NH—, —OC(O)NH—, and the like. The linking groups are bonds formedbetween components of the species of the invention, e.g., between theglycosyl moiety and the linker (L), or between the linker and themodifying group (R¹). Other exemplary linking groups are ethers,thioethers and amines. For example, in one embodiment, the linker is anamino acid residue, such as a glycine residue. The carboxylic acidmoiety of the glycine is converted to the corresponding amide byreaction with an amine on the glycosyl residue, and the amine of theglycine is converted to the corresponding amide or urethane by reactionwith an activated carboxylic acid or carbonate of the modifying group.

Another exemplary linker is a PEG moiety, e.g., a PEG moiety that isfunctionalized with an amino acid residue. The PEG linker is conjugatedto the glycosyl group through the amino acid residue at one PEG terminusand bound to R¹ through the other PEG terminus. Alternatively, the aminoacid residue is bound to R¹ and the PEG terminus, which is not bound tothe amino acid, is bound to the glycosyl group.

An exemplary species of NH-L-R¹ has the formula:—NH{C(O)(CH₂)_(a)NH}_(s){C(O)(CH₂)_(b)(OCH₂CH₂)_(c)O(CH₂)_(d)NH}_(t)R¹,in which the indices s and t are independently 0 or 1. The indices a, band d are independently integers from 0 to 20, and c is an integer from1 to 2500. Other similar linkers are based on species in which an —NHmoiety is replaced by another group, for example, —S, —O or —CH₂. Asthose of skill will appreciate one or more of the bracketed moietiescorresponding to indices s and t can be replaced with a substituted orunsubstituted alkyl or heteroalkyl moiety.

More particularly, the invention utilizes compounds in which NH-L-R¹ is:NHC(O)(CH₂)_(a)NHC(O)(CH₂)_(b)(OCH₂CH₂)_(c)O(CH₂)_(d)NHR¹,NHC(O)(CH₂)_(b)(OCH₂CH₂)_(c)O(CH₂)_(d)NHR¹,NHC(O)O(CH₂)_(b)(OCH₂CH₂)CO(CH₂)_(d)NHR¹,NH(CH₂)_(a)NHC(O)(CH₂)_(b)(OCH₂CH₂)_(c)O(CH₂)_(d)NHR¹,NHC(O)(CH₂)_(a)NHR¹, NH(CH₂)_(a)NHR¹, and NHR¹. In these formulae, theindices a, b and d are independently selected from the integers from 0to 20, preferably from 1 to 5. The index c is an integer from 1 to about2500.

In an exemplary embodiment, c is selected such that the PEG moiety isapproximately 1 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa or80 kDa.

For the purposes of convenience, the glycosyl linking groups in theremainder of this section will be based on a sialyl moiety. However, oneof skill in the art will recognize that another glycosyl moiety, such asmannosyl, galactosyl, glucosyl, or fucosyl, could be used in place ofthe sialyl moiety.

In an exemplary embodiment, the glycosyl linking group is an intactglycosyl linking group, in which the glycosyl moiety or moieties formingthe linking group are not degraded by chemical (e.g., sodiummetaperiodate) or enzymatic (e.g., oxidase) processes. Selectedconjugates of the invention include a modifying group that is attachedto the amine moiety of an amino-saccharide, e.g., mannosamine,glucosamine, galactosamine, sialic acid etc. In an exemplary embodiment,the invention provides a peptide conjugate comprising an intact glycosyllinking group having a formula that is selected from:

In Formulae I R² is H, CH₂OR⁷, COOR⁷ or OR⁷, in which R⁷ represents H,substituted or unsubstituted alkyl or substituted or unsubstitutedheteroalkyl. When COOR⁷ is a carboxylic acid or carboxylate, both formsare represented by the designation of the single structure COO⁻ or COOH.In Formulae I and II, the symbols R³, R⁴, R⁵, R⁶ and R^(6′)independently represent H, substituted or unsubstituted alkyl, OR⁸,NHC(O)R⁹. The index d is 0 or 1. R⁸ and R⁹ are independently selectedfrom H, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, sialic acid or polysialic acid. At least one of R³, R⁴, R⁵,R⁶ or R^(6′) includes a modifying group. This modifying group can be apolymeric modifying moiety e.g., PEG, linked through a bond or a linkinggroup. In an exemplary embodiment, R⁶ and R^(6′), together with thecarbon to which they are attached are components of the pyruvyl sidechain of sialic acid. In a further exemplary embodiment, the pyruvylside chain is functionalized with the polymeric modifying group. Inanother exemplary embodiment, R⁶ and R^(6′), together with the carbon towhich they are attached are components of the side chain of sialic acidand the polymeric modifying group is a component of R⁵.

Exemplary modifying group-intact glycosyl linking group cassettesaccording to this motif are based on a sialic acid structure, such asthose having the formulae:

In the formulae above, R¹ and L are as described above. Further detailabout the structure of exemplary R¹ groups is provided below.

In still a further exemplary embodiment, the conjugate is formed betweena peptide and a modified sugar in which the modifying group is attachedthrough a linker at the 6-carbon position of the modified sugar. Thus,illustrative glycosyl linking groups according to this embodiment havethe formula:

in which the radicals are as discussed above. Glycosyl linking groupsinclude, without limitation, glucose, glucosamine, N-acetyl-glucosamine,galactose, galactosamine, N-acetyl-galactosamine, mannose, mannosamine,N-acetyl-mannosamine, and the like.

In one embodiment, the present invention provides a peptide conjugatecomprising the following glycosyl linking group:

wherein D is a member selected from —OH and R¹-L-HN—; G is a memberselected from H and R¹-L- and —C(O)(C₁-C₆)alkyl; R¹ is a moietycomprising a straight-chain or branched poly(ethylene glycol) residue;and L is a linker, e.g., a bond (“zero order”), substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl. Inexemplary embodiments, when D is OH, G is R¹-L-, and when G is—C(O)(C₁-C₆)alkyl, D is R¹-L-NH—.

The invention provides a peptide conjugate that includes a glycosyllinking group having the formula:

In other embodiments, the glycosyl linking group has the formula:

in which the index t is 0 or 1.

In a still further exemplary embodiment, the glycosyl linking group hasthe formula:

in which the index t is 0 or 1.

In yet another embodiment, the glycosyl linking group has the formula:

in which the index p represents and integer from 1 to 10; and a iseither 0 or 1.

In an exemplary embodiment, a glycoPEGylated peptide conjugate of theinvention selected from the formulae set forth below:

In the formulae above, the index t is an integer from 0 to 1 and theindex p is an integer from 1 to 10. The symbol R^(15′) represents H, OH(e.g., Gal-OH), a sialyl moiety, a sialyl linking group (i.e., sialyllinking group-polymeric modifying group (Sia-L-R¹), or a sialyl moietyto which is bound a polymer modified sialyl moiety (e.g., Sia-Sia-L-R¹)(“Sia-Sia^(p)”)). Exemplary polymer modified saccharyl moieties have astructure according to Formulae I and II. An exemplary peptide conjugateof the invention will include at least one glycan having a R^(15′) thatincludes a structure according to Formulae I or II. The oxygen, with theopen valence, of Formulae I and II is preferably attached through aglycosidic linkage to a carbon of a Gal or GalNAc moiety. In a furtherexemplary embodiment, the oxygen is attached to the carbon at position 3of a galactose residue. In an exemplary embodiment, the modified sialicacid is linked α2,3-to the galactose residue. In another exemplaryembodiment, the sialic acid is linked α2,6-to the galactose residue.

In an exemplary embodiment, the sialyl linking group is a sialyl moietyto which is bound a polymer modified sialyl moiety (e.g., Sia-Sia-L-R¹)(“Sia-Sia^(p)”). Here, the glycosyl linking group is linked to agalactosyl moiety through a sialyl moiety:

An exemplary species according to this motif is prepared by conjugatingSia-L-R¹ to a terminal sialic acid of a glycan using an enzyme thatforms Sia-Sia bonds, e.g., CST-II, ST8Sia-II, ST8Sia-III and ST8Sia-IV.In some embodiments, the modified glycan is bound to the EPO peptide atone or more positions selected from Asn 24, Asn 38, Asn 83 and/or Ser126.

An exemplary species according to this motif is prepared by conjugatingSia-L-R¹ to a terminal sialic acid of the glycan at Asn 24 using anenzyme that forms Sia-Sia bonds, e.g., CST-II, ST8Sia-II, ST8Sia-III andST8Sia-IV.

In another exemplary embodiment, the glycans on the peptide conjugateshave a formula that is selected from the group:

and combinations thereof.

The glycans of this group generally correspond to those found on an EPOpeptide that is produced by insect cells (e.g., Sf-9), followed byremodeling of the glycan and glycoPEGylation according to the methodsset forth herein. For example insect-derived EPO that is expressed witha tri-mannosyl core is subsequently contacted with a GlcNAc donor and aGlcNAc transferase and a Gal donor and a Gal transferase. AppendingGlcNAc and Gal to the tri-mannosyl core is accomplished in either twosteps or a single step. A modified sialic acid is added to at least onebranch of the glycosyl moiety as discussed herein. Those Gal moietiesthat are not functionalized with the modified sialic acid are optionally“capped” by reaction with a sialic acid donor in the presence of asialyl transferase. See, FIG. 2A, FIG. 2B and FIG. 3 for exemplarystructures of glycans that include sialyl capped galactose residues, andmixtures of sialyl capped and uncapped galactose residues.

In each of the formulae above, R^(15′) is as discussed above. Moreover,an exemplary peptide conjugate of the invention will include at leastone glycan with an R¹⁵ moiety having a structure according to Formulae Ior II.

In an exemplary embodiment, at least 60% of terminal Gal moieties in apopulation of peptides is capped with sialic acid, preferably at least70%, more preferably, at least 80%, still more preferably at least 90%and even more preferably at least 95%, 96%, 97%, 98% or 99% are cappedwith sialic acid.

In another exemplary embodiment, the glycosyl linking group comprises atleast one glycosyl linking group having the formula:

wherein R¹⁵ is said sialyl linking group; and the index p is an integerselected from 1 to 10.

In an exemplary embodiment, the glycosyl linking moiety has the formula:

in which b is an integer from 0 to 1. The index s represents an integerfrom 1 to 10; and the index f represents an integer from 1 to 2500.

In certain embodiments, the EPO peptide includes three such moieties,one attached at each of Asn 24, Asn 38 and Asn 83. In anotherembodiment, the peptide includes two such moieties attached at acombination of two of these Asn moieties. There is also provided acomposition that is a mixture of these two species (i.e., PEG₃ andPEG₂). The mixture preferably includes at least 75%, preferably at least80%, more preferably at least 85%, still more preferably 90% and evenmore preferably 95%, 96%, 97% or 98% of the species that includes thethree modified glycosyl residues. Unmodified terminal Gal residues areoptionally capped with Sia as discussed above. In an exemplaryembodiment, the peptide is expressed in insect cells, remodeled andglycopegylated (FIG. 5).

In an exemplary embodiment, the polymeric modifying group is PEG. Inanother exemplary embodiment, the PEG moiety has a molecular weight ofabout 20 kDa. In another exemplary embodiment, the PEG moiety has amolecular weight of about 5 kDa. In another exemplary embodiment, thePEG moiety has a molecular weight of about 10 kDa. In another exemplaryembodiment, the PEG moiety has a molecular weight of about 40 kDa.

In an exemplary embodiment, the glycosyl linking group is a linear 10kDa-PEG-sialyl, and one or two of these glycosyl linking groups arecovalently attached to the peptide. In an exemplary embodiment, theglycosyl linking group is a linear 20 kDa-PEG-sialyl, and one or two ofthese glycosyl linking groups are covalently attached to the peptide. Inan exemplary embodiment, the glycosyl linking group is a linear 5kDa-PEG-sialyl, and one, two or three of these glycosyl linking groupsare covalently attached to the peptide. In an exemplary embodiment, theglycosyl linking group is a linear 40 kDa-PEG-sialyl, and one or two ofthese glycosyl linking groups are covalently attached to the peptide.

Modifying Groups

The peptide conjugates of the invention comprise a modifying group. Thisgroup can be covalently attached to a FGF peptide through an amino acidor a glycosyl linking group. “Modifying groups” can encompass a varietyof structures including targeting moieties, therapeutic moieties,biomolecules. Additionally, “modifying groups” include polymericmodifying groups, which are polymers which can alter a property of thepeptide such as its bioavailability or its half-life in the body.

In an exemplary embodiment, the modifying group is a targeting agentthat localizes selectively in a particular tissue due to the presence ofa targeting agent as a component of the conjugate. In an exemplaryembodiment, the targeting agent is a protein. Exemplary proteins includetransferrin (brain, blood pool), HS-glycoprotein (bone, brain, bloodpool), antibodies (brain, tissue with antibody-specific antigen, bloodpool), coagulation factors V-XII (damaged tissue, clots, cancer, bloodpool), serum proteins, e.g., α-acid glycoprotein, fetuin, α-fetalprotein (brain, blood pool), β2-glycoprotein (liver, atherosclerosisplaques, brain, blood pool), G-CSF, GM-CSF, M-CSF, and EPO (immunestimulation, cancers, blood pool, red blood cell overproduction,neuroprotection), albumin (increase in half-life), and lipoprotein E.

For the purposes of convenience, the modifying groups in the remainderof this section will be largely based on polymeric modifying groups suchas water soluble and water insoluble polymers. However, one of skill inthe art will recognize that other modifying groups, such as targetingmoieties, therapeutic moieties and biomolecules, could be used in placeof the polymeric modifying groups.

Linkers of the Modifying Groups

The linkers of the modifying group serve to attach the modifying group(ie polymeric modifying groups, targeting moieties, therapeutic moietiesand biomolecules) to the peptide. In an exemplary embodiment, thepolymeric modifying group is bound to a glycosyl linking group,generally through a heteroatom, e.g, nitrogen, on the core through alinker, L, as shown below:

R¹ is the polymeric moiety and L is selected from a bond and a linkinggroup. The index w represents an integer selected from 1-6, preferably1-3 and more preferably 1-2. Exemplary linking groups includesubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl moieties and sialic acid. An exemplary component of thelinker is an acyl moiety.

An exemplary compound according to the invention has a structureaccording to Formulae I or II above, in which at least one of R², R³,R⁴, R⁵, R⁶ or R^(6′) has the formula:

In another example according to this embodiment at least one of R², R³,R⁴, R⁵, R⁶ or R^(6′) has the formula:

in which s is an integer from 0 to 20 and R¹ is a linear polymericmodifying moiety.

In an exemplary embodiment, the polymeric modifying group-linkerconstruct is a branched structure that includes two or more polymericchains attached to central moiety. In this embodiment, the construct hasthe formula:

in which R¹ and L are as discussed above and w′ is an integer from 2 to6, preferably from 2 to 4 and more preferably from 2 to 3.

When L is a bond it is formed between a reactive functional group on aprecursor of R¹ and a reactive functional group of complementaryreactivity on the saccharyl core. When L is a non-zero order linker, aprecursor of L can be in place on the glycosyl moiety prior to reactionwith the R¹ precursor. Alternatively, the precursors of R¹ and L can beincorporated into a preformed cassette that is subsequently attached tothe glycosyl moiety. As set forth herein, the selection and preparationof precursors with appropriate reactive functional groups is within theability of those skilled in the art. Moreover, coupling the precursorsproceeds by chemistry that is well understood in the art.

In an exemplary embodiment, L is a linking group that is formed from anamino acid, or small peptide (e.g., 1-4 amino acid residues) providing amodified sugar in which the polymeric modifying group is attachedthrough a substituted alkyl linker. Exemplary linkers include glycine,lysine, serine and cysteine. The PEG moiety can be attached to the aminemoiety of the linker through an amide or urethane bond. The PEG islinked to the sulfur or oxygen atoms of cysteine and serine throughthioether or ether bonds, respectively.

In an exemplary embodiment, R⁵ includes the polymeric modifying group.In another exemplary embodiment, R⁵ includes both the polymericmodifying group and a linker, L, joining the modifying group to theremainder of the molecule. As discussed above, L can be a linear orbranched structure. Similarly, the polymeric modifying group can bebranched or linear.

Water-Soluble Polymers

Many water-soluble polymers are known to those of skill in the art andare useful in practicing the present invention. The term water-solublepolymer encompasses species such as saccharides (e.g., dextran, amylose,hyalouronic acid, poly(sialic acid), heparans, heparins, etc.);poly(amino acids), e.g., poly(aspartic acid) and poly(glutamic acid);nucleic acids; synthetic polymers (e.g., poly(acrylic acid),poly(ethers), e.g., poly(ethylene glycol)); peptides, proteins, and thelike. The present invention may be practiced with any water-solublepolymer with the sole limitation that the polymer must include a pointat which the remainder of the conjugate can be attached.

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and more WO 93/15189, and for conjugation between activatedpolymers and peptides, e.g. Coagulation Factor VIII (WO 94/15625),hemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No.4,412,989), ribonuclease and superoxide dismutase (Veronese at al., App.Biochem. Biotech. 11: 141-45 (1985)).

Exemplary water-soluble polymers are those in which a substantialproportion of the polymer molecules in a sample of the polymer are ofapproximately the same molecular weight; such polymers are“homodisperse.”

The present invention is further illustrated by reference to apoly(ethylene glycol) conjugate. Several reviews and monographs on thefunctionalization and conjugation of PEG are available. See, forexample, Harris, Macronol. Chem. Phys. C25: 325-373 (1985); Scouten,Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme Microb.Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky,Bioconjugate Chem. 6: 150-165 (1995); and Bhadra, et al., Pharmazie,57:5-29 (2002). Routes for preparing reactive PEG molecules and formingconjugates using the reactive molecules are known in the art. Forexample, U.S. Pat. No. 5,672,662 discloses a water soluble andisolatable conjugate of an active ester of a polymer acid selected fromlinear or branched poly(alkylene oxides), poly(oxyethylated polyols),poly(olefinic alcohols), and poly(acrylomorpholine).

U.S. Pat. No. 6,376,604 sets forth a method for preparing awater-soluble 1-benzotriazolylcarbonate ester of a water-soluble andnon-peptidic polymer by reacting a terminal hydroxyl of the polymer withdi(1-benzotriazoyl)carbonate in an organic solvent. The active ester isused to form conjugates with a biologically active agent such as aprotein or peptide.

WO 99/45964 describes a conjugate comprising a biologically active agentand an activated water soluble polymer comprising a polymer backbonehaving at least one terminus linked to the polymer backbone through astable linkage, wherein at least one terminus comprises a branchingmoiety having proximal reactive groups linked to the branching moiety,in which the biologically active agent is linked to at least one of theproximal reactive groups. Other branched poly(ethylene glycols) aredescribed in WO 96/21469, U.S. Pat. No. 5,932,462 describes a conjugateformed with a branched PEG molecule that includes a branched terminusthat includes reactive functional groups. The free reactive groups areavailable to react with a biologically active species, such as a proteinor peptide, forming conjugates between the poly(ethylene glycol) and thebiologically active species. U.S. Pat. No. 5,446,090 describes abifunctional PEG linker and its use in forming conjugates having apeptide at each of the PEG linker termini.

Conjugates that include degradable PEG linkages are described in WO99/34833; and WO 99/14259, as well as in U.S. Pat. No. 6,348,558. Suchdegradable linkages are applicable in the present invention.

The art-recognized methods of polymer activation set forth above are ofuse in the context of the present invention in the formation of thebranched polymers set forth herein and also for the conjugation of thesebranched polymers to other species, e.g., sugars, sugar nucleotides andthe like.

An exemplary water-soluble polymer is poly(ethylene glycol), e.g.,methoxy-poly(ethylene glycol). The poly(ethylene glycol) used in thepresent invention is not restricted to any particular form or molecularweight range. For unbranched poly(ethylene glycol) molecules themolecular weight is preferably between 500 and 100,000. A molecularweight of 2000-60,000 is preferably used and preferably of from about5,000 to about 40,000.

In other exemplary embodiments, the poly(ethylene glycol) molecule isselected from the following:

In another embodiment the poly(ethylene glycol) is a branched PEG havingmore than one PEG moiety attached. Examples of branched PEGs aredescribed in U.S. Pat. No. 5,932,462; U.S. Pat. No. 5,342,940; U.S. Pat.No. 5,643,575; U.S. Pat. No. 5,919,455; U.S. Pat. No. 6,113,906; U.S.Pat. No. 5,183,660; WO 02/09766; Kodera Y., Bioconjugate Chemistry 5:283-288 (1994); and Yamasaki et al., Agric. Biol. Chem., 52: 2125-2127,1998. In a preferred embodiment the molecular weight of eachpoly(ethylene glycol) of the branched PEG is less than or equal to40,000 daltons.

Representative polymeric modifying moieties include structures that arebased on side chain-containing amino acids, e.g., serine, cysteine,lysine, and small peptides, e.g., lys-lys. Exemplary structures include:

Those of skill will appreciate that the free amine in the di-lysinestructures can also be pegylated through an amide or urethane bond witha PEG moiety.

In yet another embodiment, the polymeric modifying moiety is a branchedPEG moiety that is based upon a tri-lysine peptide. The tri-lysine canbe mono-, di-, tri-, or tetra-PEG-ylated. Exemplary species according tothis embodiment have the formulae:

in which the indices e, f and f′ are independently selected integersfrom 1 to 2500; and the indices q, q′ and q″ are independently selectedintegers from 1 to 20.

As will be apparent to those of skill, the branched polymers of use inthe invention include variations on the themes set forth above. Forexample the di-lysine-PEG conjugate shown above can include threepolymeric subunits, the third bonded to the α-amine shown as unmodifiedin the structure above. Similarly, the use of a tri-lysinefunctionalized with three or four polymeric subunits labeled with thepolymeric modifying moiety in a desired manner is within the scope ofthe invention.

The polymeric modifying moieties can be activated for reaction with theglycosyl core. Exemplary structures of activated species (e.g.,carbonates and active esters) include:

Other activating, or leaving groups, appropriate for activating linearand branched PEGs of use in preparing the compounds set forth hereininclude, but are not limited to the species:

PEG molecules that are activated with these and other species andmethods of making the activated PEGs are set forth in WO 04/083259.

Those of skill in the art will appreciate that one or more of the m-PEGarms of the branched polymers shown above can be replaced by a PEGmoiety with a different terminus, e.g., OH, COOH, NH₂, C₂-C₁₀-alkyl,etc. Moreover, the structures above are readily modified by insertingalkyl linkers (or removing carbon atoms) between the α-carbon atom andthe functional group of the amino acid side chain. Thus, “homo”derivatives and higher homologues, as well as lower homologues arewithin the scope of cores for branched PEGs of use in the presentinvention.

The branched PEG species set forth herein are readily prepared bymethods such as that set forth in the scheme below:

in which X^(d) is O or S and r is an integer from 1 to 5. The indices eand f are independently selected integers from 1 to 2500. In anexemplary embodiment, one or both of these indices are selected suchthat the polymer is about 10 kD, 15 kD or 20 kD in molecular weight.

Thus, according to this scheme, a natural or unnatural amino acid iscontacted with an activated m-PEG derivative, in this case the tosylate,forming 1 by alkylating the side-chain heteroatom X^(d). Themono-functionalize m-PEG amino acid is submitted to N-acylationconditions with a reactive m-PEG derivative, thereby assembling branchedm-PEG 2. As one of skill will appreciate, the tosylate leaving group canbe replaced with any suitable leaving group, e.g., halogen, mesylate,triflate, etc. Similarly, the reactive carbonate utilized to acylate theamine can be replaced with an active ester, e.g., N-hydroxysuccinimide,etc., or the acid can be activated in situ using a dehydrating agentsuch as dicyclohexylcarbodiimide, carbonyldiimidazole, etc.

In other exemplary embodiments, the urea moiety is replaced by a groupsuch as a amide.

As discussed herein, the PEG of use in the conjugates of the inventioncan be linear or branched. An exemplary precursor of use to form thebranched PEG containing peptide conjugates according to this embodimentof the invention has the formula:

Another exemplary precursor of use to form the branched PEG containingpeptide conjugates according to this embodiment of the invention has theformula:

in which the indices m and n are integers independently selected from 0to 5000. A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, A¹⁰ and A¹¹ are membersindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,—NA¹²A¹³, OA¹² and —SiA¹²A¹³. A¹² and A¹³ are members independentlyselected from substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl.

The branched polymer species according to this formula are essentiallypure water-soluble polymers. X^(3′) is a moiety that includes anionizable (e.g., OH, COOH, H₂PO₄, HSO₃, NH₂, and salts thereof, etc.) orother reactive functional group, e.g., infra. C is carbon. X⁵, R¹⁶ andR¹⁷ are independently selected from non-reactive groups (e.g., H,unsubstituted alkyl, unsubstituted heteroalkyl) and polymeric arms(e.g., PEG). X² and X⁴ are linkage fragments that are preferablyessentially non-reactive under physiological conditions, which may bethe same or different. An exemplary linker includes neither aromatic norester moieties. Alternatively, these linkages can include one or moremoiety that is designed to degrade under physiologically relevantconditions, e.g., esters, disulfides, etc. X² and X⁴ join polymeric armsR¹⁶ and R¹⁷ to C. When X^(3′) is reacted with a reactive functionalgroup of complementary reactivity on a linker, sugar or linker-sugarcassette, X^(3′) is converted to a component of linkage fragment X³.

Exemplary linkage fragments for X², X³ and X⁴ are independently selectedand include S, SC(O)NH, HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH andNHC(O)O, and OC(O)NH, CH₂S, CH₂O, CH₂CH₂O, CH₂CH₂S, (CH₂)_(o)O,(CH₂)_(o)S or (CH₂)_(o)Y′-PEG wherein, Y′ is S, NH, NHC(O), C(O)NH,NHC(O)O, OC(O)NH, or O and o is an integer from 1 to 50. In an exemplaryembodiment, the linkage fragments X² and X⁴ are different linkagefragments.

In an exemplary embodiment, the precursor (Formula III), or an activatedderivative thereof, is reacted with, and thereby bound to a sugar, anactivated sugar or a sugar nucleotide through a reaction between X^(3′)and a group of complementary reactivity on the sugar moiety, e.g., anamine. Alternatively, X^(3′) reacts with a reactive functional group ona precursor to linker, L. One or more of R², R³, R⁴, R⁵, R⁶ or R^(6′) ofFormulae I and II can include the branched polymeric modifying moiety,or this moiety bound through L.

In an exemplary embodiment, the moiety:

is the linker arm, L. In this embodiment, an exemplary linker is derivedfrom a natural or unnatural amino acid, amino acid analogue or aminoacid mimetic, or a small peptide formed from one or more such species.For example, certain branched polymers found in the compounds of theinvention have the formula:

X^(a) is a linkage fragment that is formed by the reaction of a reactivefunctional group, e.g., X^(3′), on a precursor of the branched polymericmodifying moiety and a reactive functional group on the sugar moiety, ora precursor to a linker. For example, when X^(3′) is a carboxylic acid,it can be activated and bound directly to an amine group pendent from anamino-saccharide (e.g., Sia, GalNH₂, GlcNH₂, ManNH₂, etc.), forming aX^(a) that is an amide. Additional exemplary reactive functional groupsand activated precursors are described hereinbelow. The index crepresents an integer from 1 to 10. The other symbols have the sameidentity as those discussed above.

In another exemplary embodiment, X^(a) is a linking moiety formed withanother linker:

in which X^(b) is a second linkage fragment and is independentlyselected from those groups set forth for X^(a), and, similar to L, L¹ isa bond, substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl.

Exemplary species for X^(a) and X^(b) include S, SC(O)NH, HNC(O)S,SC(O)O, O, NH, NHC(O), C(O)NH and NHC(O)O, and OC(O)NH.

In another exemplary embodiment, X⁴ is a peptide bond to R¹⁷, which isan amino acid, di-peptide (e.g., Lys-Lys) or tri-peptide (e.g.,Lys-Lys-Lys) in which the alpha-amine moiety(ies) and/or side chainheteroatom(s) are modified with a polymeric modifying moiety.

In a further exemplary embodiment, the peptide conjugates of theinvention include a moiety, e.g., an R¹⁵ moiety that has a formula thatis selected from:

in which the identity of the radicals represented by the various symbolsis the same as that discussed hereinabove. La is a bond or a linker asdiscussed above for L and L¹, e.g., substituted or unsubstituted alkylor substituted or unsubstituted heteroalkyl moiety. In an exemplaryembodiment, L^(a) is a moiety of the side chain of sialic acid that isfunctionalized with the polymeric modifying moiety as shown. ExemplaryL^(a) moieties include substituted or unsubstituted alkyl chains thatinclude one or more OH or NH₂.

In yet another exemplary embodiment, the invention provides peptideconjugates having a moiety, e.g., an R¹⁵ moiety with formula:

The identity of the radicals represented by the various symbols is thesame as that discussed hereinabove. As those of skill will appreciate,the linker arm in Formulae VII and VIII is equally applicable to othermodified sugars set forth herein. In exemplary embodiment, the speciesof Formulae VII and VIII are the R¹⁵ moieties attached to the glycanstructures set forth herein.

In yet another exemplary embodiment, the EPO peptide conjugate includesa R¹⁵ moiety with a formula which is a member selected from:

in which the identities of the radicals are as discussed above. Anexemplary species for La is —CH₂)_(j)C(O)NH(CH₂)_(h)C(O)NH—, in whichthe indices h and j are independently selected integers from 0 to 10. Afurther exemplary species is —C(O)NH—. The indices m and n are integersindependently selected from 0 to 5000. A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸,A⁹, A¹⁰ and A¹¹ are members independently selected from H, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, —NA¹²A¹³, —OA¹² and —SiA¹²A¹³. A¹² and A¹³ aremembers independently selected from substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl.

In an exemplary embodiment, the glycosyl linking group has a structureaccording to the following formula:

The embodiments of the invention set forth above are further exemplifiedby reference to species in which the polymer is a water-soluble polymer,particularly poly(ethylene glycol) (“PEG”), e.g., methoxy-poly(ethyleneglycol). Those of skill will appreciate that the focus in the sectionsthat follow is for clarity of illustration and the various motifs setforth using PEG as an exemplary polymer are equally applicable tospecies in which a polymer other than PEG is utilized.

PEG of any molecular weight, e.g., 1 kDa, 2 kDa, 5 kDa, 10 kDa, 15 kDa,20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa,65 kDa, 70 kDa, 75 kDa, or 80 kDa is of use in the present invention.

In an exemplary embodiment, the R¹⁵ moiety has a formula that is amember selected from the group:

In each of the structures above, the linker fragment —NH(CH₂)_(a)— canbe present or absent.

In other exemplary embodiments, the peptide conjugate includes an R¹⁵moiety selected from the group:

In each of the formulae above, the indices e and f are independentlyselected from the integers from 1 to 2500. In further exemplaryembodiments, e and f are selected to provide a PEG moiety that is about1 kDa, 2 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, or 80 kDa.The symbol Q represents substituted or unsubstituted alkyl (e.g., C₁-C₆alkyl, e.g., methyl), substituted or unsubstituted heteroalkyl or H.

Other branched polymers have structures based on di-lysine (Lys-Lys)peptides, e.g.:

and tri-lysine peptides (Lys-Lys-Lys), e.g.:

In each of the figures above, the indices e, f, f′ and f″ representintegers independently selected from 1 to 2500. The indices q, q′ and q″represent integers independently selected from 1 to 20.

In another exemplary embodiment, the modifying group:

has a formula that is a member selected from:

wherein Q is a member selected from H and substituted or unsubstitutedC₁-C₆ alkyl. The indices e and f are integers independently selectedfrom 1 to 2500, and the index q is an integer selected from 0 to 20.

In another exemplary embodiment, the modifying group:

has a formula that is a member selected from:

wherein Q is a member selected from H and substituted or unsubstitutedC₁-C₆ alkyl. The indices e, f and f′ are integers independently selectedfrom 1 to 2500, and q and q′ are integers independently selected from 1to 20.

In another exemplary embodiment, the branched polymer has a structureaccording to the following formula:

in which the indices m and n are integers independently selected from 0to 5000. A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, A¹⁰ and A¹¹ are membersindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl, —NA²A¹³,—OA¹² and —SiA¹²A¹³. A¹² and A¹³ are members independently selected fromsubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl.

Formula IIIa is a subset of Formula III. The structures described byFormula IIIa are also encompassed by Formula III.

In another exemplary embodiment according to the formula above, thebranched polymer has a structure according to the following formula:

In an exemplary embodiment, A¹ and A² are each —OCH₃ or H.

In an illustrative embodiment, the modified sugar is sialic acid andselected modified sugar compounds of use in the invention have theformulae:

The indices a, b and d are integers from 0 to 20. The index c is aninteger from 1 to 2500. The structures set forth above can be componentsof R¹⁵.

In another illustrative embodiment, a primary hydroxyl moiety of thesugar is functionalized with the modifying group. For example, the9-hydroxyl of sialic acid can be converted to the corresponding amineand functionalized to provide a compound according to the invention.Formulae according to this embodiment include:

The structures set forth above can be components of R¹⁵.

In a further exemplary embodiment, the invention provides modifiedsugars in which the 6-hydroxyl position is converted to thecorresponding amine moiety, which bears a linker-modifying groupcassette such as those set forth above. Exemplary saccharyl groups thatcan be used as the core of these modified sugars include Gal, GalNAc,Glc, GlcNAc, Fuc, Xyl, Man, and the like. A representative modifiedsugar according to this embodiment has the formula:

in which R³-R⁵ and R⁷ are members independently selected from H, OH,C(O)CH₃, NH, and NHC(O)CH₃. R⁶ is OR¹, NHR¹ or L-R¹, which is asdescribed above.

Although the present invention is exemplified in the preceding sectionsby reference to PEG, as those of skill will appreciate, an array ofpolymeric modifying moieties is of use in the compounds and methods setforth herein.

In selected embodiments, R¹ or L-R¹ is a branched PEG, for example, oneof the species set forth above. In an exemplary embodiment, the branchedPEG structure is based on a cysteine peptide. Illustrative modifiedsugars according to this embodiment include:

in which X⁴ is a bond or O. In each of the structures above, thealkylamine linker —(CH₂)_(a)NH— can be present or absent. The structuresset forth above can be components of R¹⁵/R^(15′).

As discussed herein, the polymer-modified sialic acids of use in theinvention may also be linear structures. Thus, the invention providesfor conjugates that include a sialic acid moiety derived from astructure such as:

in which the indices q and e are as discussed above.

Exemplary modified sugars are modified with water-soluble orwater-insoluble polymers. Examples of useful polymer are furtherexemplified below.

In another exemplary embodiment, the peptide is derived from insectcells, remodeled by adding GlcNAc and Gal to the mannose core andglycopegylated using a sialic acid bearing a linear PEG moiety,affording a EPO peptide that comprises at least one moiety having theformula:

in which the index t is an integer from 0 to 1; the index s representsan integer from 1 to 10; and the index f represents an integer from 1 to2500.Water-Insoluble Polymers

In another embodiment, analogous to those discussed above, the modifiedsugars include a water-insoluble polymer, rather than a water-solublepolymer. The conjugates of the invention may also include one or morewater-insoluble polymers. This embodiment of the invention isillustrated by the use of the conjugate as a vehicle with which todeliver a therapeutic peptide in a controlled manner. Polymeric drugdelivery systems are known in the art. See, for example, Dunn et al.,Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium SeriesVol. 469, American Chemical Society, Washington, D.C. 1991. Those ofskill in the art will appreciate that substantially any known drugdelivery system is applicable to the conjugates of the presentinvention.

The motifs forth above for R¹, L-R¹, R¹⁵, R^(15′) and other radicals areequally applicable to water-insoluble polymers, which may beincorporated into the linear and branched structures without limitationutilizing chemistry readily accessible to those of skill in the art.

Representative water-insoluble polymers include, but are not limited to,polyphosphazines, poly(vinyl alcohols), polyamides, polycarbonates,polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkyleneoxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes, poly(methyl methacrylate), poly(ethyl methacrylate),poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate),poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate)polyethylene, polypropylene, poly(ethylene glycol), poly(ethyleneoxide), poly (ethylene terephthalate), poly(vinyl acetate), polyvinylchloride, polystyrene, polyvinyl pyrrolidone, pluronics andpolyvinylphenol and copolymers thereof.

Synthetically modified natural polymers of use in conjugates of theinvention include, but are not limited to, alkyl celluloses,hydroxyalkyl celluloses, cellulose ethers, cellulose esters, andnitrocelluloses. Particularly preferred members of the broad classes ofsynthetically modified natural polymers include, but are not limited to,methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, celluloseacetate, cellulose propionate, cellulose acetate butyrate, celluloseacetate phthalate, carboxymethyl cellulose, cellulose triacetate,cellulose sulfate sodium salt, and polymers of acrylic and methacrylicesters and alginic acid.

These and the other polymers discussed herein can be readily obtainedfrom commercial sources such as Sigma Chemical Co. (St. Louis, Mo.),Polysciences (Warrenton, Pa.), Aldrich (Milwaukee, Wis.), Fluka(Ronkonkoma, N.Y.), and BioRad (Richmond, Calif.), or else synthesizedfrom monomers obtained from these suppliers using standard techniques.

Representative biodegradable polymers of use in the conjugates of theinvention include, but are not limited to, polylactides, polyglycolidesand copolymers thereof, poly(ethylene terephthalate), poly(butyricacid), poly(valeric acid), poly(lactide-co-caprolactone),poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, blends andcopolymers thereof. Of particular use are compositions that form gels,such as those including collagen, pluronics and the like.

The polymers of use in the invention include “hybrid’ polymers thatinclude water-insoluble materials having within at least a portion oftheir structure, a bioresorbable molecule. An example of such a polymeris one that includes a water-insoluble copolymer, which has abioresorbable region, a hydrophilic region and a plurality ofcrosslinkable functional groups per polymer chain.

For purposes of the present invention, “water-insoluble materials”includes materials that are substantially insoluble in water orwater-containing environments. Thus, although certain regions orsegments of the copolymer may be hydrophilic or even water-soluble, thepolymer molecule, as a whole, does not to any substantial measuredissolve in water.

For purposes of the present invention, the term “bioresorbable molecule”includes a region that is capable of being metabolized or broken downand resorbed and/or eliminated through normal excretory routes by thebody. Such metabolites or break down products are preferablysubstantially non-toxic to the body.

The bioresorbable region may be either hydrophobic or hydrophilic, solong as the copolymer composition as a whole is not renderedwater-soluble. Thus, the bioresorbable region is selected based on thepreference that the polymer, as a whole, remains water-insoluble.Accordingly, the relative properties, i.e., the kinds of functionalgroups contained by, and the relative proportions of the bioresorbableregion, and the hydrophilic region are selected to ensure that usefulbioresorbable compositions remain water-insoluble.

Exemplary resorbable polymers include, for example, syntheticallyproduced resorbable block copolymers of poly(α-hydroxy-carboxylicacid)/poly(oxyalkylene, (see, Cohn et al., U.S. Pat. No. 4,826,945).These copolymers are not crosslinked and are water-soluble so that thebody can excrete the degraded block copolymer compositions. See, Youneset al., J Biomed Mater. Res. 21: 1301-1316 (1987); and Cohn et al., JBiomed. Mater. Res. 22: 993-1009 (1988).

Presently preferred bioresorbable polymers include one or morecomponents selected from poly(esters), poly(hydroxy acids),poly(lactones), poly(amides), poly(ester-amides), poly(amino acids),poly(anhydrides), poly(orthoesters), poly(carbonates),poly(phosphazines), poly(phosphoesters), poly(thioesters),polysaccharides and mixtures thereof. More preferably still, thebiosresorbable polymer includes a poly(hydroxy) acid component. Of thepoly(hydroxy) acids, polylactic acid, polyglycolic acid, polycaproicacid, polybutyric acid, polyvaleric acid and copolymers and mixturesthereof are preferred.

In addition to forming fragments that are absorbed in vivo(“bioresorbed”), preferred polymeric coatings for use in the methods ofthe invention can also form an excretable and/or metabolizable fragment.

Higher order copolymers can also be used in the present invention. Forexample, Casey et al., U.S. Pat. No. 4,438,253, which issued on Mar. 20,1984, discloses tri-block copolymers produced from thetransesterification of poly(glycolic acid) and an hydroxyl-endedpoly(alkylene glycol). Such compositions are disclosed for use asresorbable monofilament sutures. The flexibility of such compositions iscontrolled by the incorporation of an aromatic orthocarbonate, such astetra-p-tolyl orthocarbonate into the copolymer structure.

Other polymers based on lactic and/or glycolic acids can also beutilized. For example, Spinu, U.S. Pat. No. 5,202,413, which issued onApr. 13, 1993, discloses biodegradable multi-block copolymers havingsequentially ordered blocks of polylactide and/or polyglycolide producedby ring-opening polymerization of lactide and/or glycolide onto eitheran oligomeric diol or a diamine residue followed by chain extension witha di-functional compound, such as, a diisocyanate, diacylchloride ordichlorosilane.

Bioresorbable regions of coatings useful in the present invention can bedesigned to be hydrolytically and/or enzymatically cleavable. Forpurposes of the present invention, “hydrolytically cleavable” refers tothe susceptibility of the copolymer, especially the bioresorbableregion, to hydrolysis in water or a water-containing environment.Similarly, “enzymatically cleavable” as used herein refers to thesusceptibility of the copolymer, especially the bioresorbable region, tocleavage by endogenous or exogenous enzymes.

When placed within the body, the hydrophilic region can be processedinto excretable and/or metabolizable fragments. Thus, the hydrophilicregion can include, for example, polyethers, polyalkylene oxides,polyols, poly(vinyl pyrrolidine), poly(vinyl alcohol), poly(alkyloxazolines), polysaccharides, carbohydrates, peptides, proteins andcopolymers and mixtures thereof. Furthermore, the hydrophilic region canalso be, for example, a poly(alkylene) oxide. Such poly(alkylene) oxidescan include, for example, poly(ethylene) oxide, poly(propylene) oxideand mixtures and copolymers thereof.

Polymers that are components of hydrogels are also useful in the presentinvention. Hydrogels are polymeric materials that are capable ofabsorbing relatively large quantities of water. Examples of hydrogelforming compounds include, but are not limited to, polyacrylic acids,sodium carboxymethylcellulose, polyvinyl alcohol, polyvinyl pyrrolidine,gelatin, carrageenan and other polysaccharides,hydroxyethylenemethacrylic acid (HEMA), as well as derivatives thereof,and the like. Hydrogels can be produced that are stable, biodegradableand bioresorbable. Moreover, hydrogel compositions can include subunitsthat exhibit one or more of these properties.

Bio-compatible hydrogel compositions whose integrity can be controlledthrough crosslinking are known and are presently preferred for use inthe methods of the invention. For example, Hubbell et al., U.S. Pat. No.5,410,016, which issued on Apr. 25, 1995 and U.S. Pat. No. 5,529,914,which issued on Jun. 25, 1996, disclose water-soluble systems, which arecrosslinked block copolymers having a water-soluble central blocksegment sandwiched between two hydrolytically labile extensions. Suchcopolymers are further end-capped with photopolymerizable acrylatefunctionalities. When crosslinked, these systems become hydrogels. Thewater soluble central block of such copolymers can include poly(ethyleneglycol); whereas, the hydrolytically labile extensions can be apoly(α-hydroxy acid), such as polyglycolic acid or polylactic acid. See,Sawhney et al., Macromolecules 26: 581-587 (1993).

In another preferred embodiment, the gel is a thermoreversible gel.Thermoreversible gels including components, such as pluronics, collagen,gelatin, hyalouronic acid, polysaccharides, polyurethane hydrogel,polyurethane-urea hydrogel and combinations thereof are presentlypreferred.

In yet another exemplary embodiment, the conjugate of the inventionincludes a component of a liposome. Liposomes can be prepared accordingto methods known to those skilled in the art, for example, as describedin Eppstein et al., U.S. Pat. No. 4,522,811. For example, liposomeformulations may be prepared by dissolving appropriate lipid(s) (such asstearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline,arachadoyl phosphatidyl choline, and cholesterol) in an inorganicsolvent that is then evaporated, leaving behind a thin film of driedlipid on the surface of the container. An aqueous solution of the activecompound or its pharmaceutically acceptable salt is then introduced intothe container. The container is then swirled by hand to free lipidmaterial from the sides of the container and to disperse lipidaggregates, thereby forming the liposomal suspension.

The above-recited microparticles and methods of preparing themicroparticles are offered by way of example and they are not intendedto define the scope of microparticles of use in the present invention.It will be apparent to those of skill in the art that an array ofmicroparticles, fabricated by different methods, is of use in thepresent invention.

The structural formats discussed above in the context of thewater-soluble polymers, both straight-chain and branched are generallyapplicable with respect to the water-insoluble polymers as well. Thus,for example, the cysteine, serine, dilysine, and trilysine branchingcores can be functionalized with two water-insoluble polymer moieties.The methods used to produce these species are generally closelyanalogous to those used to produce the water-soluble polymers.

The Methods

In addition to the conjugates discussed above, the present inventionprovides methods for preparing these and other conjugates. Moreover, theinvention provides methods of preventing, curing or ameliorating adisease state by administering a conjugate of the invention to a subjectat risk of developing the disease or a subject that has the disease.

In exemplary embodiments, the conjugate is formed between a polymericmodifying moiety, a therapeutic moiety, targeting moiety or abiomolecule and a glycosylated or non-glycosylated peptide. The polymeris conjugated to the peptide via a glycosyl linking group, which isinterposed between, and covalently linked to both the peptide (orglycosyl residue) and the modifying group (e.g., water-soluble polymer).The method includes contacting the peptide with a mixture containing amodified sugar and an enzyme, e.g., a glycosyltransferase thatconjugates the modified sugar to the substrate. The reaction isconducted under conditions appropriate to form a covalent bond betweenthe modified sugar and the peptide. The sugar moiety of the modifiedsugar is preferably selected from nucleotide sugars.

In an exemplary embodiment, the modified sugar, such as those set forthabove, is activated as the corresponding nucleotide sugars. Exemplarysugar nucleotides that are used in the present invention in theirmodified form include nucleotide mono-, di- or triphosphates or analogsthereof. In a preferred embodiment, the modified sugar nucleotide isselected from a UDP-glycoside, CMP-glycoside, or a GDP-glycoside. Evenmore preferably, the sugar nucleotide portion of the modified sugarnucleotide is selected from UDP-galactose, UDP-galactosamine,UDP-glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid,or CMP-NeuAc. In an exemplary embodiment, the nucleotide phosphate isattached to C-1.

Thus, in an illustrative embodiment in which the glycosyl moiety issialic acid, the method of the invention utilizes compounds having theformulae:

in which L-R¹ is as discussed above, and L¹-R¹ represents a linker boundto the modifying group. As with L, exemplary linker species according toL¹ include a bond, alkyl or heteroalkyl moieties.

Moreover, as discussed above, the present invention provides for the useof nucleotide sugars that are modified with a water-soluble polymer,which is either straight-chain or branched. For example, compoundshaving the formula shown below are of use to prepare conjugates withinthe scope of the present invention:

in which X⁴ is O or a bond.

The invention also provides for the use of sugar nucleotides modifiedwith L-R¹ at the 6-carbon position. Exemplary species according to thisembodiment include:

in which the R groups, and L, represent moieties as discussed above. Theindex “y” is 0, 1 or 2. In an exemplary embodiment, L is a bond betweenNH and R¹. The base is a nucleic acid base.

Exemplary nucleotide sugars of use in the invention in which the carbonat the 6-position is modified include species having the stereochemistryof GDP mannose, e.g.:

in which X⁵ is a bond or O. The index i represents 0 or 1. The index arepresents an integer from 1 to 20. The indices e and f independentlyrepresent integers from 1 to 2500. Q, as discussed above, is H orsubstituted or unsubstituted C₁-C₆ alkyl. As those of skill willappreciate, the serine derivative, in which S is replaced with O alsofalls within this general motif.

In a still further exemplary embodiment, the invention provides aconjugate in which the modified sugar is based on the stereochemistry ofUDP galactose. An exemplary nucleotide sugar of use in this inventionhas the structure:

In another exemplary embodiment, the nucleotide sugar is based on thestereochemistry of glucose. Exemplary species according to thisembodiment have the formulae:

In general, the sugar moiety or sugar moiety-linker cassette and the PEGor PEG-linker cassette groups are linked together through the use ofreactive groups, which are typically transformed by the linking processinto a new organic functional group or unreactive species. The sugarreactive functional group(s), is located at any position on the sugarmoiety. Reactive groups and classes of reactions useful in practicingthe present invention are generally those that are well known in the artof bioconjugate chemistry. Currently favored classes of reactionsavailable with reactive sugar moieties are those, which proceed underrelatively mild conditions. These include, but are not limited tonucleophilic substitutions (e.g., reactions of amines and alcohols withacyl halides, active esters), electrophilic substitutions (e.g., enaminereactions) and additions to carbon-carbon and carbon-heteroatom multiplebonds (e.g., Michael reaction, Diels-Alder addition). These and otheruseful reactions are discussed in, for example, March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982.

Useful reactive functional groups pendent from a sugar nucleus ormodifying group include, but are not limited to:

-   -   (a) carboxyl groups and various derivatives thereof including,        but not limited to, N-hydroxysuccinimide esters,        N-hydroxybenztriazole esters, acid halides, acyl imidazoles,        thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and        aromatic esters;    -   (b) hydroxyl groups, which can be converted to, e.g., esters,        ethers, aldehydes, etc.    -   (c) haloalkyl groups, wherein the halide can be later displaced        with a nucleophilic group such as, for example, an amine, a        carboxylate anion, thiol anion, carbanion, or an alkoxide ion,        thereby resulting in the covalent attachment of a new group at        the functional group of the halogen atom;    -   (d) dienophile groups, which are capable of participating in        Diels-Alder reactions such as, for example, maleimido groups;    -   (e) aldehyde or ketone groups, such that subsequent        derivatization is possible via formation of carbonyl derivatives        such as, for example, imines, hydrazones, semicarbazones or        oximes, or via such mechanisms as Grignard addition or        alkyllithium addition;    -   (f) sulfonyl halide groups for subsequent reaction with amines,        for example, to form sulfonamides;    -   (g) thiol groups, which can be, for example, converted to        disulfides or reacted with acyl halides;    -   (h) amine or sulfhydryl groups, which can be, for example,        acylated, alkylated or oxidized;    -   (i) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc; and    -   (j) epoxides, which can react with, for example, amines and        hydroxyl compounds.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assemblethe reactive sugar nucleus or modifying group. Alternatively, a reactivefunctional group can be protected from participating in the reaction bythe presence of a protecting group. Those of skill in the art understandhow to protect a particular functional group such that it does notinterfere with a chosen set of reaction conditions. For examples ofuseful protecting groups, see, for example, Greene et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

Modified Sugars

The present invention uses modified sugars and modified sugarnucleotides to form conjugates of the modified sugars. In modified sugarcompounds of use in the invention, the sugar moiety is preferably asaccharide, a deoxy-saccharide, an amino-saccharide, or an N-acylsaccharide. The term “saccharide” and its equivalents, “saccharyl,”“sugar,” and “glycosyl” refer to monomers, dimers, oligomers andpolymers. The sugar moiety is also functionalized with a modifyinggroup. The modifying group is conjugated to the sugar moiety, typically,through conjugation with an amine, sulfhydryl or hydroxyl, e.g., primaryhydroxyl, moiety on the sugar. In an exemplary embodiment, the modifyinggroup is attached through an amine moiety on the sugar, e.g., through anamide, a urethane or a urea that is formed through the reaction of theamine with a reactive derivative of the modifying group.

Any sugar can be utilized as the sugar core of the glycosyl linkinggroup of the conjugates of the invention. Exemplary sugar cores that areuseful in forming the compositions of the invention include, but are notlimited to, glucose, galactose, mannose, fucose, and sialic acid. Otheruseful sugars include amino sugars such as glucosamine, galactosamine,mannosamine, the 5-amine analogue of sialic acid and the like. The sugarcore can be a structure found in nature or it can be modified to providea site for conjugating the modifying group.

In the discussion that follows, a number of specific examples ofmodified sugars that are useful in practicing the present invention areset forth. In the exemplary embodiments, a sialic acid derivative isutilized as the sugar nucleus to which the modifying group is attached.The focus of the discussion on sialic acid derivatives is for clarity ofillustration only and should not be construed to limit the scope of theinvention. Those of skill in the art will appreciate that a variety ofother sugar moieties can be activated and derivatized in a manneranalogous to that set forth using sialic acid as an example. Forexample, numerous methods are available for modifying galactose,glucose, N-acetylgalactosamine and fucose to name a few sugarsubstrates, which are readily modified by art recognized methods. See,for example, Elhalabi et al., Curr. Med. Chem. 6: 93 (1999); and Schaferet al., J. Org. Chem. 65: 24 (2000)).

In an exemplary embodiment, the modified sugar is based upon a6-amino-N-acetyl-glycosyl moiety. As shown below forN-acetylgalactosamine, the 6-amino-sugar moiety is readily prepared bystandard methods.

In the scheme above, the index n represents an integer from 1 to 2500.In an exemplary embodiment, this index is selected such that the polymeris about 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa,50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa or 80 kDa in molecularweight. The symbol “A” represents an activating group, e.g., a halo, acomponent of an activated ester (e.g., a N-hydroxysuccinimide ester), acomponent of a carbonate (e.g., p-nitrophenyl carbonate) and the like.Those of skill in the art will appreciate that other PEG-amidenucleotide sugars are readily prepared by this and analogous methods.

The acceptor peptide is typically synthesized de novo, or recombinantlyexpressed in a prokaryotic cell (e.g., bacterial cell, such as E. coli)or in a eukaryotic cell such as a mammalian, yeast, insect, fungal orplant cell. The peptide can be either a full-length protein or afragment. Moreover, the peptide can be a wild type or mutated peptide.In an exemplary embodiment, the peptide includes a mutation that addsone or more N- or O-linked glycosylation sites to the peptide sequence.

The method of the invention also provides for modification ofincompletely glycosylated peptides that are produced recombinantly. Manyrecombinantly produced glycoproteins are incompletely glycosylated,exposing carbohydrate residues that may have undesirable properties,e.g., immunogenicity, recognition by the RES. Employing a modified sugarin a method of the invention, the peptide can be simultaneously furtherglycosylated and derivatized with, e.g., a water-soluble polymer,therapeutic agent, or the like. The sugar moiety of the modified sugarcan be the residue that would properly be conjugated to the acceptor ina fully glycosylated peptide, or another sugar moiety with desirableproperties.

Those of skill will appreciate that the invention can be practiced usingsubstantially any peptide or glycopeptide from any source. Exemplarypeptides with which the invention can be practiced are set forth in WO03/031464, and the references set forth therein.

Peptides modified by the methods of the invention can be synthetic orwild-type peptides or they can be mutated peptides, produced by methodsknown in the art, such as site-directed mutagenesis. Glycosylation ofpeptides is typically either N-linked or O-linked. An exemplaryN-linkage is the attachment of the modified sugar to the side chain ofan asparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of a carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. O-linked glycosylation refers to the attachment ofone sugar (e.g., N-acetylgalactosamine, galactose, mannose, GlcNAc,glucose, fucose or xylose) to the hydroxy side chain of a hydroxyaminoacid, preferably serine or threonine, although unusual or non-naturalamino acids, e.g., 5-hydroxyproline or 5-hydroxylysine may also be used.

Moreover, in addition to peptides, the methods of the present inventioncan be practiced with other biological structures (e.g., glycolipids,lipids, sphingoids, ceramides, whole cells, and the like, containing aglycosylation site).

Addition of glycosylation sites to a peptide or other structure isconveniently accomplished by altering the amino acid sequence such thatit contains one or more glycosylation sites. The addition may also bemade by the incorporation of one or more species presenting an —OHgroup, preferably serine or threonine residues, within the sequence ofthe peptide (for O-linked glycosylation sites). The addition may be madeby mutation or by full chemical synthesis of the peptide. The peptideamino acid sequence is preferably altered through changes at the DNAlevel, particularly by mutating the DNA encoding the peptide atpreselected bases such that codons are generated that will translateinto the desired amino acids. The DNA mutation(s) are preferably madeusing methods known in the art.

In an exemplary embodiment, the glycosylation site is added by shufflingpolynucleotides. Polynucleotides encoding a candidate peptide can bemodulated with DNA shuffling protocols. DNA shuffling is a process ofrecursive recombination and mutation, performed by random fragmentationof a pool of related genes, followed by reassembly of the fragments by apolymerase chain reaction-like process. See, e.g., Stemmer, Proc. Natl.Acad. Sci. USA 91:10747-10751 (1994); Stemmer, Nature 370:389-391(1994); and U.S. Pat. Nos. 5,605,793, 5,837,458, 5,830,721 and5,811,238.

Exemplary peptides with which the present invention can be practiced,methods of adding or removing glycosylation sites, and adding orremoving glycosyl structures or substructures are described in detail inWO03/031464 and related U.S. and PCT applications.

The present invention also takes advantage of adding to (or removingfrom) a peptide one or more selected glycosyl residues, after which amodified sugar is conjugated to at least one of the selected glycosylresidues of the peptide. The present embodiment is useful, for example,when it is desired to conjugate the modified sugar to a selectedglycosyl residue that is either not present on a peptide or is notpresent in a desired amount. Thus, prior to coupling a modified sugar toa peptide, the selected glycosyl residue is conjugated to the peptide byenzymatic or chemical coupling. In another embodiment, the glycosylationpattern of a glycopeptide is altered prior to the conjugation of themodified sugar by the removal of a carbohydrate residue from theglycopeptide. See, for example WO 98/31826.

Addition or removal of any carbohydrate moieties present on theglycopeptide is accomplished either chemically or enzymatically. Anexemplary chemical deglycosylation is brought about by exposure of thepolypeptide variant to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the peptide intact. Chemicaldeglycosylation is described by Hakimuddin et al., Arch. Biochem.Biophys. 259: 52 (1987) and by Edge et al., Anal. Biochem. 118: 131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptidevariants can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol. 138:350 (1987).

In an exemplary embodiment, the peptide is essentially completelydesialylated with neuraminidase prior to performing glycoconjugation orremodeling steps on the peptide. Following the glycoconjugation orremodeling, the peptide is optionally re-sialylated using asialyltransferase. In an exemplary embodiment, the re-sialylation occursat essentially each (e.g., >80%, preferably greater than 85%, greaterthan 90%, preferably greater than 95% and more preferably greater than96%, 97%, 98% or 99%) terminal saccharyl acceptor in a population ofsialyl acceptors. In a preferred embodiment, the saccharide has asubstantially uniform sialylation pattern (i.e., substantially uniformglycosylation pattern).

Chemical addition of glycosyl moieties is carried out by anyart-recognized method. Enzymatic addition of sugar moieties ispreferably achieved using a modification of the methods set forthherein, substituting native glycosyl units for the modified sugars usedin the invention. Other methods of adding sugar moieties are disclosedin U.S. Pat. Nos. 5,876,980, 6,030,815, 5,728,554, and 5,922,577.

Exemplary attachment points for selected glycosyl residue include, butare not limited to: (a) consensus sites for N-linked glycosylation, andsites for O-linked glycosylation; (b) terminal glycosyl moieties thatare acceptors for a glycosyltransferase; (c) arginine, asparagine andhistidine; (d) free carboxyl groups; (e) free sulfhydryl groups such asthose of cysteine; (f) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (g) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (h) the amide group ofglutamine. Exemplary methods of use in the present invention aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston, CRC CRIT. REV. BIOCHEM., pp. 259-306 (1981).

In one embodiment, the invention provides a method for linking two ormore peptides through a linking group. The linking group is of anyuseful structure and may be selected from straight- and branched-chainstructures. Preferably, each terminus of the linker, which is attachedto a peptide, includes a modified sugar (i.e., a nascent intact glycosyllinking group).

In an exemplary method of the invention, two peptides are linkedtogether via a linker moiety that includes a polymeric (e.g., PEGlinker). The construct conforms to the general structure set forth inthe cartoon above. As described herein, the construct of the inventionincludes two intact glycosyl linking groups (i.e., s+t=1). The focus ona PEG linker that includes two glycosyl groups is for purposes ofclarity and should not be interpreted as limiting the identity of linkerarms of use in this embodiment of the invention.

Thus, a PEG moiety is functionalized at a first terminus with a firstglycosyl unit and at a second terminus with a second glycosyl unit. Thefirst and second glycosyl units are preferably substrates for differenttransferases, allowing orthogonal attachment of the first and secondpeptides to the first and second glycosyl units, respectively. Inpractice, the (glycosyl)¹-PEG-(glycosyl)² linker is contacted with thefirst peptide and a first transferase for which the first glycosyl unitis a substrate, thereby forming (peptide)¹-(glycosyl)¹-PEG-(glycosyl)².Transferase and/or unreacted peptide is then optionally removed from thereaction mixture. The second peptide and a second transferase for whichthe second glycosyl unit is a substrate are added to the(peptide)¹-(glycosyl)¹-PEG-(glycosyl)² conjugate, forming(peptide)¹-(glycosyl)¹-PEG-(glycosyl)²-(peptide)²; at least one of theglycosyl residues is either directly or indirectly O-linked. Those ofskill in the art will appreciate that the method outlined above is alsoapplicable to forming conjugates between more than two peptides by, forexample, the use of a branched PEG, dendrimer, poly(amino acid),polysaccharide or the like.

In an exemplary embodiment, the peptide that is modified by a method ofthe invention is a glycopeptide that is produced in mammalian cells(e.g., CHO cells) or in a transgenic animal and thus, contains N- and/orO-linked oligosaccharide chains, which are incompletely sialylated. Theoligosaccharide chains of the glycopeptide lacking a sialic acid andcontaining a terminal galactose residue can be PEGylated, PPGylated orotherwise modified with a modified sialic acid.

In Scheme 1, the amino glycoside 1, is treated with the active ester ofa protected amino acid (e.g., glycine) derivative, converting the sugaramine residue into the corresponding protected amino acid amide adduct.The adduct is treated with an aldolase to form α-hydroxy carboxylate 2.Compound 2 is converted to the corresponding CMP derivative by theaction of CMP-SA synthetase, followed by catalytic hydrogenation of theCMP derivative to produce compound 3. The amine introduced via formationof the glycine adduct is utilized as a locus of PEG attachment byreacting compound 3 with an activated PEG or PPG derivative (e.g.,PEG-C(O)NHS, PEG-OC(O)O-p-nitrophenyl), producing species such as 4 or5, respectively.

Conjugation of Modified Sugars to Peptides

The PEG modified sugars are conjugated to a glycosylated ornon-glycosylated peptide using an appropriate enzyme to mediate theconjugation. Preferably, the concentrations of the modified donorsugar(s), enzyme(s) and acceptor peptide(s) are selected such thatglycosylation proceeds until the acceptor is consumed. Theconsiderations discussed below, while set forth in the context of asialyltransferase, are generally applicable to other glycosyltransferasereactions.

A number of methods of using glycosyltransferases to synthesize desiredoligosaccharide structures are known and are generally applicable to theinstant invention. Exemplary methods are described, for instance, WO96/32491, Ito et al., Pure Appl. Chem. 65: 753 (1993), U.S. Pat. Nos.5,352,670, 5,374,541, 5,545,553, commonly owned U.S. Pat. Nos.6,399,336, and 6,440,703, and commonly owned published PCT applications,WO 03/031464, WO 04/033651, WO 04/099231, which are incorporated hereinby reference.

The present invention is practiced using a single glycosyltransferase ora combination of glycosyltransferases. For example, one can use acombination of a sialyltransferase and a galactosyltransferase. In thoseembodiments using more than one enzyme, the enzymes and substrates arepreferably combined in an initial reaction mixture, or the enzymes andreagents for a second enzymatic reaction are added to the reactionmedium once the first enzymatic reaction is complete or nearly complete.By conducting two enzymatic reactions in sequence in a single vessel,overall yields are improved over procedures in which an intermediatespecies is isolated. Moreover, cleanup and disposal of extra solventsand by-products is reduced.

In a preferred embodiment, each of the first and second enzyme is aglycosyltransferase. In another preferred embodiment, one enzyme is anendoglycosidase. In an additional preferred embodiment, more than twoenzymes are used to assemble the modified glycoprotein of the invention.The enzymes are used to alter a saccharide structure on the peptide atany point either before or after the addition of the modified sugar tothe peptide.

In another embodiment, the method makes use of one or more exo- orendoglycosidase. The glycosidase is typically a mutant, which isengineered to form glycosyl bonds rather than rupture them. The mutantglycanase typically includes a substitution of an amino acid residue foran active site acidic amino acid residue. For example, when theendoglycanase is endo-H, the substituted active site residues willtypically be Asp at position 130, Glu at position 132 or a combinationthereof. The amino acids are generally replaced with serine, alanine,asparagine, or glutamine.

The mutant enzyme catalyzes the reaction, usually by a synthesis stepthat is analogous to the reverse reaction of the endoglycanasehydrolysis step. In these embodiments, the glycosyl donor molecule(e.g., a desired oligo- or mono-saccharide structure) contains a leavinggroup and the reaction proceeds with the addition of the donor moleculeto a GlcNAc residue on the protein. For example, the leaving group canbe a halogen, such as fluoride. In other embodiments, the leaving groupis a Asn, or a Asn-peptide moiety. In further embodiments, the GlcNAcresidue on the glycosyl donor molecule is modified. For example, theGlcNAc residue may comprise a 1,2 oxazoline moiety.

In a preferred embodiment, each of the enzymes utilized to produce aconjugate of the invention are present in a catalytic amount. Thecatalytic amount of a particular enzyme varies according to theconcentration of that enzyme's substrate as well as to reactionconditions such as temperature, time and pH value. Means for determiningthe catalytic amount for a given enzyme under preselected substrateconcentrations and reaction conditions are well known to those of skillin the art.

The temperature at which an above process is carried out can range fromjust above freezing to the temperature at which the most sensitiveenzyme denatures. Preferred temperature ranges are about 0° C. to about55° C., and more preferably about 20° C. to about 37° C., e.g. about 30°C. In another exemplary embodiment, one or more components of thepresent method are conducted at an elevated temperature using athermophilic enzyme.

The reaction mixture is maintained for a period of time sufficient forthe acceptor to be glycosylated, thereby forming the desired conjugate.Some of the conjugate can often be detected after a few h, withrecoverable amounts usually being obtained within 24 h or less. Those ofskill in the art understand that the rate of reaction is dependent on anumber of variable factors (e.g, enzyme concentration, donorconcentration, acceptor concentration, temperature, solvent volume),which are optimized for a selected system.

The present invention also provides for the industrial-scale productionof modified peptides. As used herein, an industrial scale generallyproduces at least one gram of finished, purified conjugate.

In the discussion that follows, the invention is exemplified by theconjugation of modified sialic acid moieties to a glycosylated peptide.The exemplary modified sialic acid is labeled with PEG. The focus of thefollowing discussion on the use of PEG-modified sialic acid andglycosylated peptides is for clarity of illustration and is not intendedto imply that the invention is limited to the conjugation of these twopartners. One of skill understands that the discussion is generallyapplicable to the additions of modified glycosyl moieties other thansialic acid. Moreover, the discussion is equally applicable to themodification of a glycosyl unit with agents other than PEG includingother PEG moieties, therapeutic moieties, and biomolecules.

An enzymatic approach can be used for the selective introduction ofPEGylated or PPGylated carbohydrates onto a peptide or glycopeptide. Themethod utilizes modified sugars containing PEG, PPG, or a maskedreactive functional group, and is combined with the appropriateglycosyltransferase or glycosynthase. By selecting theglycosyltransferase that will make the desired carbohydrate linkage andutilizing the modified sugar as the donor substrate, the PEG or PPG canbe introduced directly onto the peptide backbone, onto existing sugarresidues of a glycopeptide or onto sugar residues that have been addedto a peptide.

In an exemplary embodiment, an acceptor for a sialyltransferase ispresent on the peptide to be modified either as a naturally occurringstructure or it is placed there recombinantly, enzymatically orchemically. Suitable acceptors, include, for example, galactosylacceptors such as Galβ1,4GlcNAc, Galβ1,4GalNAc, Galβ1,3GalNAc,lacto-N-tetraose, Galβ1,3GlcNAc, Galβ1,3Ara, Galβ1,6GlcNAc, Galβ1,4Glc(lactose), and other acceptors known to those of skill in the art (see,e.g., Paulson et al., J. Biol. Chem. 253: 5617-5624 (1978)). Exemplarysialyltransferases are set forth herein, see, e.g., FIG. 9.

In one embodiment, an acceptor for the sialyltransferase is present onthe glycopeptide to be modified upon in vivo synthesis of theglycopeptide. Such glycopeptides can be sialylated using the claimedmethods without prior modification of the glycosylation pattern of theglycopeptide. Alternatively, the methods of the invention can be used tosialylate a peptide that does not include a suitable acceptor; one firstmodifies the peptide to include an acceptor by methods known to those ofskill in the art. In an exemplary embodiment, a GalNAc residue is addedby the action of a GalNAc transferase.

In an exemplary embodiment, the galactosyl acceptor is assembled byattaching a galactose residue to an appropriate acceptor linked to thepeptide, e.g., a GlcNAc. The method includes incubating the peptide tobe modified with a reaction mixture that contains a suitable amount of agalactosyltransferase (e.g., Galβ1,3 or Galβ1,4), and a suitablegalactosyl donor (e.g., UDP-galactose). The reaction is allowed toproceed substantially to completion or, alternatively, the reaction isterminated when a preselected amount of the galactose residue is added.Other methods of assembling a selected saccharide acceptor will beapparent to those of skill in the art.

In yet another embodiment, glycopeptide-linked oligosaccharides arefirst “trimmed,” either in whole or in part, to expose either anacceptor for the sialyltransferase or a moiety to which one or moreappropriate residues can be added to obtain a suitable acceptor. Enzymessuch as glycosyltransferases and endoglycosidases (see, for example U.S.Pat. No. 5,716,812) are useful for the attaching and trimming reactions.In another embodiment of this method, the sialic acid moieties of thepeptide are essentially completely removed (e.g., at least 90, at least95 or at least 99%), exposing an acceptor for a modified sialic acid.

In the discussion that follows, the method of the invention isexemplified by the use of modified sugars having a PEG moiety attachedthereto. The focus of the discussion is for clarity of illustration.Those of skill will appreciate that the discussion is equally relevantto those embodiments in which the modified sugar bears a therapeuticmoiety, biomolecule or the like.

In an exemplary embodiment of the invention in which a carbohydrateresidue is “trimmed” prior to the addition of the modified sugar highmannose is trimmed back to the first generation biantennary structure. Amodified sugar bearing a PEG moiety is conjugated to one or more of thesugar residues exposed by the “trimming back.” In one example, a PEGmoiety is added via a GlcNAc moiety conjugated to the PEG moiety. Themodified GlcNAc is attached to one or both of the terminal mannoseresidues of the biantennary structure. Alternatively, an unmodifiedGlcNAc can be added to one or both of the termini of the branchedspecies.

In another exemplary embodiment, a PEG moiety is added to one or both ofthe terminal mannose residues of the biantennary structure via amodified sugar having a galactose residue, which is conjugated to aGlcNAc residue added onto the terminal mannose residues. Alternatively,an unmodified Gal can be added to one or both terminal GlcNAc residues.

In yet a further example, a PEG moiety is added onto a Gal residue usinga modified sialic acid such as those discussed above.

In another exemplary embodiment, a high mannose structure is “trimmedback” to the mannose from which the biantennary structure branches. Inone example, a PEG moiety is added via a GlcNAc modified with thepolymer. Alternatively, an unmodified GlcNAc is added to the mannose,followed by a Gal with an attached PEG moiety. In yet anotherembodiment, unmodified GlcNAc and Gal residues are sequentially added tothe mannose, followed by a sialic acid moiety modified with a PEGmoiety.

A high mannose structure can also be trimmed back to the elementarytri-mannosyl core.

In a further exemplary embodiment, high mannose is “trimmed back” to theGlcNAc to which the first mannose is attached. The GlcNAc is conjugatedto a Gal residue bearing a PEG moiety. Alternatively, an unmodified Galis added to the GlcNAc, followed by the addition of a sialic acidmodified with a water-soluble sugar. In yet a further example, theterminal GlcNAc is conjugated with Gal and the GlcNAc is subsequentlyfucosylated with a modified fucose bearing a PEG moiety.

High mannose may also be trimmed back to the first GlcNAc attached tothe Asn of the peptide. In one example, the GlcNAc of theGlcNAc-(Fuc)_(a) residue is conjugated wit ha GlcNAc bearing a watersoluble polymer. In another example, the GlcNAc of the GlcNAc-(Fuc)_(a)residue is modified with Gal, which bears a water soluble polymer. In astill further embodiment, the GlcNAc is modified with Gal, followed byconjugation to the Gal of a sialic acid modified with a PEG moiety.

Other exemplary embodiments are set forth in commonly owned U.S. Patentapplication Publications: 20040132640; 20040063911; 20040137557; U.S.patent application Ser. Nos. 10/369,979; 10/410,913; 10/360,770;10/410,945 and PCT/US02/32263 each of which is incorporated herein byreference.

The Examples set forth above provide an illustration of the power of themethods set forth herein. Using the methods described herein, it ispossible to “trim back” and build up a carbohydrate residue ofsubstantially any desired structure. The modified sugar can be added tothe termini of the carbohydrate moiety as set forth above, or it can beintermediate between the peptide core and the terminus of thecarbohydrate.

In an exemplary embodiment, an existing sialic acid is removed from aglycopeptide using a sialidase, thereby unmasking all or most of theunderlying galactosyl residues. Alternatively, a peptide or glycopeptideis labeled with galactose residues, or an oligosaccharide residue thatterminates in a galactose unit. Following the exposure of or addition ofthe galactose residues, an appropriate sialyltransferase is used to adda modified sialic acid.

In another exemplary embodiment, an enzyme that transfers sialic acidonto sialic acid is utilized. This method can be practiced withouttreating a sialylated glycan with a sialidase to expose glycan residuesbeneath the sialic acid. An exemplary polymer-modified sialic acid is asialic acid modified with poly(ethylene glycol). Other exemplary enzymesthat add sialic acid and modified sialic acid moieties onto sialic acidresidues of a glycan or exchange an existing sialic acid residue on aglycan for these species include ST3Gal3, CST-II, ST8Sia-II, ST8Sia-IIIand ST8Sia-IV.

In yet a further approach, a masked reactive functionality is present onthe sialic acid. The masked reactive group is preferably unaffected bythe conditions used to attach the modified sialic acid to theerythropoietin. After the covalent attachment of the modified sialicacid to the peptide, the mask is removed and the peptide is conjugatedwith an agent such as PEG. The agent is conjugated to the peptide in aspecific manner by its reaction with the unmasked reactive group on themodified sugar residue.

Any modified sugar can be used with its appropriate glycosyltransferase,depending on the terminal sugars of the oligosaccharide side chains ofthe glycopeptide. As discussed above, the terminal sugar of theglycopeptide required for introduction of the PEGylated structure can beintroduced naturally during expression or it can be produced postexpression using the appropriate glycosidase(s), glycosyltransferase(s)or mix of glycosidase(s) and glycosyltransferase(s).

In a further exemplary embodiment, UDP-galactose-PEG is reacted withβ1,4-galactosyltransferase, thereby transferring the modified galactoseto the appropriate terminal N-acetylglucosamine structure. The terminalGlcNAc residues on the glycopeptide may be produced during expression,as may occur in such expression systems as mammalian, insect, plant orfungus, but also can be produced by treating the glycopeptide with asialidase and/or glycosidase and/or glycosyltransferase, as required.

In another exemplary embodiment, a GlcNAc transferase, such as GNT1-5,is utilized to transfer PEGylated-GlcNAc to a terminal mannose residueon a glycopeptide. In a still further exemplary embodiment, an the N-and/or O-linked glycan structures are enzymatically removed from aglycopeptide to expose an amino acid or a terminal glycosyl residue thatis subsequently conjugated with the modified sugar. For example, anendoglycanase is used to remove the N-linked structures of aglycopeptide to expose a terminal GlcNAc as a GlcNAc-linked-Asn on theglycopeptide. UDP-Gal-PEG and the appropriate galactosyltransferase isused to introduce the PEG-galactose functionality onto the exposedGlcNAc.

In an alternative embodiment, the modified sugar is added directly tothe peptide backbone using a glycosyltransferase known to transfer sugarresidues to the peptide backbone. Exemplary glycosyltransferases usefulin practicing the present invention include, but are not limited to,GalNAc transferases (GalNAc T1-14), GlcNAc transferases,fucosyltransferases, glucosyltransferases, xylosyltransferases,mannosyltransferases and the like. Use of this approach allows thedirect addition of modified sugars onto peptides that lack anycarbohydrates or, alternatively, onto existing glycopeptides. In bothcases, the addition of the modified sugar occurs at specific positionson the peptide backbone as defined by the substrate specificity of theglycosyltransferase and not in a random manner as occurs duringmodification of a protein's peptide backbone using chemical methods. Anarray of agents can be introduced into proteins or glycopeptides thatlack the glycosyltransferase substrate peptide sequence by engineeringthe appropriate amino acid sequence into the polypeptide chain.

In each of the exemplary embodiments set forth above, one or moreadditional chemical or enzymatic modification steps can be utilizedfollowing the conjugation of the modified sugar to the peptide. In anexemplary embodiment, an enzyme (e.g., fucosyltransferase) is used toappend a glycosyl unit (e.g., fucose) onto the terminal modified sugarattached to the peptide. In another example, an enzymatic reaction isutilized to “cap” sites to which the modified sugar failed to conjugate.Alternatively, a chemical reaction is utilized to alter the structure ofthe conjugated modified sugar. For example, the conjugated modifiedsugar is reacted with agents that stabilize or destabilize its linkagewith the peptide component to which the modified sugar is attached. Inanother example, a component of the modified sugar is deprotectedfollowing its conjugation to the peptide. One of skill will appreciatethat there is an array of enzymatic and chemical procedures that areuseful in the methods of the invention at a stage after the modifiedsugar is conjugated to the peptide. Further elaboration of the modifiedsugar-peptide conjugate is within the scope of the invention.

Enzymes and reaction conditions for preparing the conjugates of thepresent invention are discussed in detail in the parent of the instantapplication as well as co-owned published PCT patent applications WO03/031464, WO 04/033651, WO 04/099231.

In a selected embodiment, set forth in Example 2, an EPO peptide,expressed in insect cells, is remodeled such that glycans on theremodeled glycopeptide include a GlcNAc-Gal glycosyl residue. Theaddition of GlcNAc and Gal can occur as separate reactions or as asingle reaction in a single vessel. In this example, GlcNAc-transferaseI and Gal-transferase I are used. The modified sialyl moiety is addedusing ST3Gal-III.

In another embodiment, as illustrated in Example 3, the addition ofGlcNAc, Gal and modified Sia can also occur in a single reaction vessel,using the enzymes set forth above. Example 4 sets forth a method inwhich each of the enzymatic remodeling and glycoPEGylation steps arecarried out individually.

When the peptide is expressed in mammalian cells, different methods areof use. In Example 5, the peptide is conjugated without need forremodeling prior to conjugation by contacting the peptide with asialyltransferase that transfers the modified sialic acid directly ontoa sialic acid on the peptide forming Sia-Sia-L-R¹, or exchanges a sialicacid on the peptide for the modified sialic acid, forming Sia-L-R¹. Anexemplary enzyme of use in this method is CST-II. Other enzymes that addsialic acid to sialic acid are known to those of skill in the art andexamples of such enzymes are set forth in FIG. 9.

Another method of preparing the conjugates of the invention is set forthin Example 6. The peptide expressed in a mammalian system isdesialylated using a sialidase. The exposed Gal residue is sialylatedwith a modified sialic acid using a sialyltransferase specific forO-linked glycans, providing an EPO peptide with an O-linked modifiedglycan. The desialylated, modified EPO peptide is optionally partiallyor fully re-sialylated by using a sialyltransferase such as ST3GalIII.

In another aspect, the invention provides a method of making a PEGylatederythropoietin of the invention. The method includes: (a) contacting asubstrate erythropoietin peptide comprising a glycosyl group selectedfrom:

with a PEG-sialic acid donor having the formula:

and an enzyme that transfers PEG-sialic acid from said donor onto amember selected from the Gal and the Sia of said glycosyl group, underconditions appropriate for said transfer. An exemplary modified sialicacid donor is CMP-sialic acid modified, through a linker moiety, with apolymer, e.g., a straight chain or branched poly(ethylene glycol)moiety.

In an exemplary embodiment, the PEG-sialic acid donor has the formula:

In another exemplary emodiment, the PEG-sialic acid donor h as theformula:

In a further exemplary embodiment, the EPO peptide is expressed in anappropriate expression system prior to being glycopegylated orremodeled. Exemplary expression systems include Sf-9/baculovirus andChinese Hamster Ovary (CHO) cells.

In an exemplary embodiment, the invention provides a method of makingGlycoPEGylated EPO in which the pH is between 5.5 and 8.0. In anotherexemplary embodiment, the invention provides a method of makingGlycoPEGylated EPO in which metals, i.e. MnCl₂ or MgCl₂, are not presentin the reaction mixture. In yet another exemplary embodiment, theinvention provides a method of making GlycoPEGylated EPO in which areacted nucleotide sugar, i.e. CMP, GDP, are not present in the reactionmixture. In still another exemplary embodiment, the invention provides amethod of making GlycoPEGylated EPO in which a chelating agent, i.e.EDTA, is not present in the reaction mixture.

In a further exemplary embodiment, the total amount of PEGylated sugaris added to the reaction mixture at one time. In another exemplaryembodiment, the total amount of PEGylated sugar is added sequentially.

Purification of Erythropoietin Conjugates

The products produced by the above processes can be used withoutpurification. However, it is usually preferred to recover the productand one or more of the intermediates, e.g., nucleotide sugars, branchedand linear PEG species, modified sugars and modified nucleotide sugars.Standard, well-known techniques for recovery of glycosylated saccharidessuch as thin or thick layer chromatography, column chromatography, ionexchange chromatography, or membrane filtration can be used. It ispreferred to use membrane filtration, more preferably utilizing areverse osmotic membrane, or one or more column chromatographictechniques for the recovery as is discussed hereinafter and in theliterature cited herein. For instance, membrane filtration wherein themembranes have molecular weight cutoff of about 3000 to about 10,000 canbe used to remove proteins such as glycosyl transferases. Nanofiltrationor reverse osmosis can then be used to remove salts and/or purify theproduct saccharides (see, e.g., WO 98/15581). Nanofilter membranes are aclass of reverse osmosis membranes that pass monovalent salts but retainpolyvalent salts and uncharged solutes larger than about 100 to about2,000 Daltons, depending upon the membrane used. Thus, in a typicalapplication, saccharides prepared by the methods of the presentinvention will be retained in the membrane and contaminating salts willpass through.

If the peptide is produced intracellularly, as a first step, theparticulate debris, either host cells or lysed fragments, is removed.Following glycoPEGylation, the PEGylated peptide is purified byart-recognized methods, for example, by centrifugation orultrafiltration; optionally, the protein may be concentrated with acommercially available protein concentration filter, followed byseparating the polypeptide variant from other impurities by one or moresteps selected from immunoaffinity chromatography, ion-exchange columnfractionation (e.g., on diethylaminoethyl (DEAE) or matrices containingcarboxymethyl or sulfopropyl groups), chromatography on Blue-Sepharose,CM Blue-Sepharose, MONO-Q, MONO-S, lentil lectin-Sepharose,WGA-Sepharose, Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, PhenylToyopearl, or protein A Sepharose, SDS-PAGE chromatography, silicachromatography, chromatofocusing, reverse phase HPLC (e.g., silica gelwith appended aliphatic groups), gel filtration using, e.g., Sephadexmolecular sieve or size-exclusion chromatography, chromatography oncolumns that selectively bind the polypeptide, and ethanol or ammoniumsulfate precipitation.

Modified glycopeptides produced in culture are usually isolated byinitial extraction from cells, enzymes, etc., followed by one or moreconcentration, salting-out, aqueous ion-exchange, or size-exclusionchromatography steps. Additionally, the modified glycoprotein may bepurified by affinity chromatography. Finally, HPLC may be employed forfinal purification steps.

A protease inhibitor, e.g., methylsulfonylfluoride (PMSF) may beincluded in any of the foregoing steps to inhibit proteolysis andantibiotics or preservatives may be included to prevent the growth ofadventitious contaminants.

Within another embodiment, supernatants from systems which produce themodified glycopeptide of the invention are first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate may be applied to a suitablepurification matrix. For example, a suitable affinity matrix maycomprise a ligand for the peptide, a lectin or antibody molecule boundto a suitable support. Alternatively, an anion-exchange resin may beemployed, for example, a matrix or substrate having pendant DEAE groups.Suitable matrices include acrylamide, agarose, dextran, cellulose, orother types commonly employed in protein purification. Alternatively, acation-exchange step may be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are particularly preferred.

Other methods of use in purification include size exclusionchromatography (SEC), hydroxyapatite chromatography, hydrophobicinteraction chromatography and chromatography on Blue Sepharose. Theseand other useful methods are illustrated in co-assigned U.S. ProvisionalPatent No. (Attorney Docket No. 40853-01-5168-P1, filed May 6, 2005).

One or more RP-HPLC steps employing hydrophobic RP-HPLC media, e.g.,silica gel having pendant methyl or other aliphatic groups, may beemployed to further purify a polypeptide conjugate composition. Some orall of the foregoing purification steps, in various combinations, canalso be employed to provide a homogeneous or essentially homogeneousmodified glycoprotein.

The modified glycopeptide of the invention resulting from a large-scalefermentation may be purified by methods analogous to those disclosed byUrdal et al., J. Chromatog. 296:171 (1984). This reference describes twosequential, RP-HPLC steps for purification of recombinant human IL-2 ona preparative HPLC column. Alternatively, techniques such as affinitychromatography may be utilized to purify the modified glycoprotein.

In an exemplary embodiment, the purification is accomplished by themethods set forth in commonly owned, co-assigned U.S. Provisional PatentNo. (Attorney Docket No. 40853-01-5168-P1, filed May 6, 2005).

Pharmaceutical Compositions

In another aspect, the invention provides a pharmaceutical composition.The pharmaceutical composition includes a pharmaceutically acceptablediluent and a covalent conjugate between a non-naturally-occurring, PEGmoiety, therapeutic moiety or biomolecule and a glycosylated ornon-glycosylated peptide. The polymer, therapeutic moiety or biomoleculeis conjugated to the peptide via an intact glycosyl linking groupinterposed between and covalently linked to both the peptide and thepolymer, therapeutic moiety or biomolecule.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Suitable formulations for use in thepresent invention are found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249:1527-1533(1990).

The pharmaceutical compositions may be formulated for any appropriatemanner of administration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268 and 5,075,109.

Commonly, the pharmaceutical compositions are administered parenterally,e.g., intravenously. Thus, the invention provides compositions forparenteral administration which comprise the compound dissolved orsuspended in an acceptable carrier, preferably an aqueous carrier, e.g.,water, buffered water, saline, PBS and the like. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents and thelike.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably from 5 to 9 and most preferably from 7 and 8.

In some embodiments the glycopeptides of the invention can beincorporated into liposomes formed from standard vesicle-forming lipids.A variety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The targeting of liposomesusing a variety of targeting agents (e.g., the sialyl galactosides ofthe invention) is well known in the art (see, e.g., U.S. Pat. Nos.4,957,773 and 4,603,044).

Standard methods for coupling targeting agents to liposomes can be used.These methods generally involve incorporation into liposomes of lipidcomponents, such as phosphatidylethanolamine, which can be activated forattachment of targeting agents, or derivatized lipophilic compounds,such as lipid-derivatized glycopeptides of the invention.

Targeting mechanisms generally require that the targeting agents bepositioned on the surface of the liposome in such a manner that thetarget moieties are available for interaction with the target, forexample, a cell surface receptor. The carbohydrates of the invention maybe attached to a lipid molecule before the liposome is formed usingmethods known to those of skill in the art (e.g., alkylation oracylation of a hydroxyl group present on the carbohydrate with a longchain alkyl halide or with a fatty acid, respectively). Alternatively,the liposome may be fashioned in such a way that a connector portion isfirst incorporated into the membrane at the time of forming themembrane. The connector portion must have a lipophilic portion, which isfirmly embedded and anchored in the membrane. It must also have areactive portion, which is chemically available on the aqueous surfaceof the liposome. The reactive portion is selected so that it will bechemically suitable to form a stable chemical bond with the targetingagent or carbohydrate, which is added later. In some cases it ispossible to attach the target agent to the connector molecule directly,but in most instances it is more suitable to use a third molecule to actas a chemical bridge, thus linking the connector molecule which is inthe membrane with the target agent or carbohydrate which is extended,three dimensionally, off of the vesicle surface.

The compounds prepared by the methods of the invention may also find useas diagnostic reagents. For example, labeled compounds can be used tolocate areas of inflammation or tumor metastasis in a patient suspectedof having an inflammation. For this use, the compounds can be labeledwith ¹²⁵I, ¹⁴C, or tritium.

The active ingredient used in the pharmaceutical compositions of thepresent invention is glycoPEGylated erythropoietin and its derivativeshaving the biological properties of causing bone marrow cells toincrease production of reticulocytes and red blood cells.

The formulation of the present invention is useful as a parenteralformulation in treating blood disorders characterized by low ordefective red blood cell production such as various forms of anemia,including anemias associated with chronic renal failure, zidovidinetreated HIV infected patients, and cancer patients on chemotherapy. Itmay also have application in the treatment of a variety of diseasestates, disorders and states of hematologic irregularity such as sicklecell disease, beta-thalassemia, cystic fibrosis, pregnancy and menstrualdisorders, early anemia of prematurity, spinal cord injury, spaceflight, acute blood loss, aging and the like. Preferably, the EPOcomposition of the present invention is administered parenterally (e.g.IV, IM, SC or IP). Effective dosages are expected to vary considerablydepending on the condition being treated and the route of administrationbut are expected to be in the range of about 0.1 (˜7 U) to 100 (˜7000 U)μg/kg body weight of the active material. Preferable doses for treatmentof anemic conditions are about 50 to about 300 Units/kg three times aweek. Because the present invention provides an erythropoietin with anenhanced in vivo residence time, the stated dosages are optionallylowered when a composition of the invention is administered.

In another embodiment, the invention provides a method of treating atissue injury in a subject in need thereof. Exemplary injuries includethose characterized by damage resulting from ischemia, trauma,inflammation or contact with toxic substances. The method includes thestep of administering to the subject an amount of a polymer-modifiederythropoietin peptide of the invention effective to ameliorate thetissue injury in the subject. An exemplary class of protection ortreatment includes neuroprotection (e.g., treatment of stroke,Alzheimer's, Parkinson's and other degenerative neurological disorders).Methods of using EPO for tissue protection are known in the art. See forexample, U.S. Pat. No. 6,531,121. The modified EPO of the invention isalso of use in treating patients with diseases such as compromisedkidney function, cancer, and retinopathy. In a further exemplaryembodiment, the EPO peptide of use in the methods isnon-erythropoietically or essentially non-erythropoietically activepeptide.

Preparative methods for species of use in preparing the compositions ofthe invention are generally set forth in various patent publications,e.g., US 20040137557; WO 04/083258; and WO 04/033651. The followingexamples are provided to illustrate the conjugates, and methods and ofthe present invention, but not to limit the claimed invention.

Erythropoietin Formulations

An aspect of the invention provides an erythropoietin (EPO) formulationincluding a polymer-modified EPO peptide of the invention. Usually theEPO formulation of the invention contains from about 0.01 mg/ml to about100 mg/ml, 0.1 mg/ml to about 10 mg/ml, or 0.1 mg/ml to about 1 mg/ml ofthe polymer-modified EPO peptide of the invention, e.g., based on EPOpeptide concentration. Alternatively, the EPO formulation of theinvention contains at least about 10 units, 50 units, or 100 units/μg ofpolymer-modified EPO peptide of the invention or at least about 1000units, 2000 units, or 5000 units/μg of EPO peptide

In one embodiment, the EPO formulation of the invention is substantiallystable, e.g., has minimum or non-detectable level of aggregation asmeasured by SDS-PAGE or size exclusion chromatography (SEC). Forexample, the EPO formulation of the invention does not have anydetectable level of aggregation, as measured by SEC, for up to one, two,or three months at 5° C., 18° C., or up to room temperature, e.g., 30°C. Alternatively, the EPO formulation of the invention has less than25%, 22%, 20%, 15%, 10%, 8%, 5%, 3%, 2.5%, or 1.5% aggregation, asmeasured by SEC, for up to three weeks, four weeks, or five weeks at 40°C.

According to the present invention, the stability of the EPO formulationcan also be determined by the distribution of tri-+tetra-PEGylated EPOpeptide in the formulation, i.e., where the modified sialic acid moietycomprising the PEG moiety, is attached to three or four branches of theglycosyl residues on the EPO peptide in the formulation. In oneembodiment, the EPO formulation of the invention has less than 20%, 15%,10%, 5%, 2%, 1% decrease of its tri-+tetra-PEG-EPO content, as measuredby RP-HPLC, for up to three weeks, four weeks, five weeks, two months,or three months at 5° C., 18° C., or up to room temperature, e.g., 30°C. In another embodiment, the EPO formulation of the invention has nosubstantial decrease, e.g., less than 1%, 0.5%, 0.1%, 0.05%, or 0%decrease of its tri-+tetra-PEG-EPO content, as measured by RP-HPLC, forup to three months at 5° C.

Usually the stability of the EPO formulation of the invention isassociated with the concentration of the polymer-modified EPO peptide ofthe invention. The higher the concentration of polymer-modified EPOpeptide is in a formulation, the higher the level of aggregationbecomes. Aggregation of the polymer-modified EPO peptide can bediminished by controlling the pH, e.g. at about 6.5. In one embodiment,when the concentration of EPO peptide in the formulation is equal to orless than 0.2 mg/ml the EPO formulation of the invention does not haveany detectable level of aggregation, as measured by SEC, for up to threemonths at 5° C. or up to room temperature, e.g., 30° C., oralternatively the EPO formulation of the invention has less than 3%,2.6%, 1.6% or 1.5% aggregation, as measured by SEC, for up to five weeksat 40° C. In another embodiment, when the concentration of EPO peptidein the formulation is equal to or less than 0.2 mg/ml the EPOformulation of the invention has less than 8%, 6.8%, 6%, 5%, 4%, 2.7%,1.6%, 1%, 0.9%, 0.5%, 0.1%, or 0% decrease of its tri-+tetra-PEG-EPOcontent, as measured by RP-HPLC, for up to five weeks or three months at5° C., room temperature, e.g., 30° C. or 40° C.

In yet another embodiment, when the concentration of EPO peptide in theformulation is equal to or less than 0.5 mg/ml the EPO formulation ofthe invention does not have any detectable level of aggregation, asmeasured by SEC, for up to three months at 5° C., or alternatively theEPO formulation of the invention has less than 25%, 22%, 15%, 10%, 9%,or 8% aggregation, as measured by SEC, for up to five weeks at 40° C. Instill another embodiment, when the concentration of EPO peptide in theformulation is equal to or less than 0.5 mg/ml the EPO formulation ofthe invention has less than 15%, 12%, 11.1%, 5.8%, 5.7%, 6.2%, 1.7%,1.2%, 0.6%, 0.1%, or 0% decrease of its tri-+tetra-PEG-EPO content, asmeasured by RP-HPLC, for up to five weeks or three months at 5° C., roomtemperature, e.g., 30° C. or 40° C.

The stability of the EPO formulation can also be related to the pH rangeof the EPO formulation. In one embodiment, the EPO formulation of theinvention has less than 2.6%, 1.6%, or 1.5% aggregation, as measured bySEC, for up to five weeks at 40° C. when the pH of EPO formulation is5.5, 6, or 6.5, respectively. In another embodiment, the EPO formulationof the invention has less than 4.6%, 1.8%, or 0.9% aggregation, asmeasured by SEC, for up to three months at 30° C. when the pH of EPOformulation is 5.5, 6, or 6.5, respectively. In yet another embodiment,the EPO formulation of the invention has less than 22.2%, 9.8%, or 8.1%aggregation, as measured by SEC, for up to five weeks at 40° C. when thepH of EPO formulation is 5.5, 6, or 6.5, respectively. In still yetanother embodiment, the EPO formulation of the invention has nodetectable level of aggregation, as measured by SEC, for up to threemonths at 5° C. or room temperature, e.g., 30° C. when the pH of EPOformulation is from about 5.5 to about 6.5, e.g., pH 5.5, 6, or 6.5.

In still another embodiment, the EPO formulation of the invention hasless than 6.8%, 2.7%, or 1.6% decrease of its tri-+tetra-PEG-EPOcontent, as measured by RP-HPLC, for up to five weeks at 40° C. when thepH of EPO formulation is 5.5, 6, or 6.5, respectively. In yet anotherembodiment, the EPO formulation of the invention has no substantialdecrease, e.g., less than 1%, 0.5%, 0.1%, 0.05%, or 0% decrease of itstri-+tetra-PEG-EPO content, as measured by RP-HPLC, for up to threemonths at 5° C. when the pH of the EPO formulation is from about 5.5 toabout 6.5, e.g., pH5.5, 6, or 6.5. In yet another embodiment, the EPOformulation of the invention has less than 6%, 0.9%, or 0% decrease ofits tri-+tetra-PEG-EPO content, as measured by RP-HPLC, for up to threemonths at room temperature, e.g., 30° C. when the pH of EPO formulationis 5.5, 6, or 6.5, respectively.

In still another embodiment, the EPO formulation of the invention hasless than 11.1%, 5.8%, or 5.7% decrease of its tri-+tetra-PEG-EPOcontent, as measured by RP-HPLC, for up to five weeks at 40° C. when thepH of EPO formulation is 5.5, 6, or 6.5, respectively. In yet anotherembodiment, the EPO formulation of the invention has no substantialdecrease, e.g., less than 1%, 0.5%, 0.1%, 0.05% or 0% decrease of itstri-+tetra-PEG-EPO content, as measured by RP-HPLC, for up to threemonths at 5° C. when the pH of the EPO formulation is from about 5.5 toabout 6.5, e.g., pH 5.5, 6, or 6.5. In yet another embodiment, the EPOformulation of the invention has less than 6.2%, 1.7%, or 1.2% decreaseof its tri-+tetra-PEG-EPO content, as measured by RP-HPLC, for up tothree months at room temperature, e.g., 30° C. when the pH of the EPOformulation is 5.5, 6, or 6.5, respectively.

According to another embodiment of the invention, the EPO formulation ofthe invention has a pH range from about 4 to about 8, from about 5 toabout 7 or from about 5.5 to about 6.5. Generally, the pH range of theEPO formulation of the invention is associated with the buffer systemused for the formulation, but as will be discussed, the pH range willalso be affected by a variety of other components in the formulation,e.g. tonicity adjusting agents. In one embodiment, the pH range of theEPO formulation is about 5.5 when acetate buffer is used for theformulation. In another embodiment, the pH range of the EPO formulationis from about 6 to about 6.5 when citrate buffer is used.

According to yet another embodiment of the invention, the EPOformulation of the invention contains a buffer, e.g., from about 5 mM toabout 1M, from about 5 mM to about 100 mM, from about 5 mM to about 50mM, from about 100 mM to about 200 mM or from about 5 mM to about 20 mM.In general, the buffer selected for the preparation of the EPOformulation of the invention can comprise one or more of any number ofsalts including, but not limited to acetate, carbonate, citrate,gluconate, histidine, lactate, maleate, phosphate, succinate, tartrateand tris. In particular, formulations comprising citrate buffer and asurfactant have desirable characteristics such as superior stabilityagainst aggregation and hydrolysis. In some embodiments, the EPOformulation of the invention includes from about 1 mM to about 50 mM ofcitrate buffer and more preferably from about 5 mM to about 25 mM ofcitrate buffer. Exemplary EPO formulations include about 10 mM ofcitrate buffer. Formulations comprising succinate are also useful inthat they tend to be less highly oxidized than citrate formulations.

In one embodiment, the buffer for the preparation of the EPO formulationis acetate, citrate, phosphate or succinate for a pH range from about 4to about 8. In another embodiment, the buffer for the EPO formulation ofthe invention is acetate, citrate, histidine, phosphate or succinate fora pH range from about 5.5 to about 6.5. In yet another embodiment, thebuffer in the EPO formulation of the invention is sodium acetate orsodium citrate at a concentration from about 5 mM to about 20 mM. Any ofthe buffers disclosed herein can be used in the EPO formulations eithersingly or in combination.

According to yet another embodiment of the invention, the EPOformulation of the invention includes one or more tonicity adjustingagent, e.g., adjusts the tonicity of the formulation to be isotonic. Inone embodiment, the tonicity adjusting agent suitable for the EPOformulation of the invention can be any suitable agent including withoutlimitation, sodium chloride, sodium sulfate, ammonium sulfate, sodiumphosphate, sucrose, trehalose, sorbitol, mannitol, histidine, arginine,and glycine. In another embodiment, the tonicity adjusting agent for theEPO formulation of the invention is sodium chloride, e.g., from about 70mM to about 150 mM. In yet another embodiment, the tonicity adjustingagent for the EPO formulation of the invention is sucrose, e.g. fromgreater than 0 mM to about 300 mM, from about 65 mM to about 120 mM. Inexemplary embodiments of the invention, both sodium chloride and sucroseare used as tonicity adjusting agents for their synergistic effect onthe stability of the EPO formulation.

According to yet another embodiment of the invention, the EPOformulation of the invention includes one or more surfactants, e.g.,from about 0.001% to 1%, from about 0.01% to about 0.5%, or from about0.005% to about 0.01%. In one embodiment, surfactants such as SDS,polysorbates or pluronics are used in the EPO formulation of theinvention. In another embodiment, polysorbate 20 (also known asTween-20), e.g., from about 0.005% to about 0.01% is used in the EPOformulation of the invention. Formulations comprising Tween-20 and abuffer have particuarly desirable characteristics and demonstratesuperior stability in terms of their aggregation and hydrolysis rates.In some embodiments of the present invention, about 0.001% to about 0.1%of Tween-20 is used. More preferably, about 0.005% to about 0.05% ofTween-20 is used in the EPO formulation of the invention, and inexemplary embodiments, about 0.01% of Tween-20 is used. In yet anotherembodiment, the EPO formulation of the invention includes PEG or PEGderivatives as surfactants in amounts ranging from about 1% to about20%.

According to yet another embodiment of the invention, the EPOformulation of the invention includes at least one stabilizer.Stabilizers can stabilize the folded state of the protein through avariety of mechanisms known in the art, e.g. by preferential hydration,by masking the hydrophobic patch of the unfolded species, by inhibitingthe ionic interactions between molecules, by binding to the proteinmolecules, or by preventing oxidation of protein molecules duringexposure to air/liquid interface. Examples of stabilizers that can beused in embodiments of the invention include disaccharides, amino acids,glycerol, albumin or PEG. Exemplary amino acids of interest includeglycine, lysine, histidine, and arginine. In some embodiments of theinvention, about 10 mM to about 500 mM of amino acids are used. Any ofthese stabilizers can be used in the EPO formulation either singly or incombination.

According to yet another embodiment of the invention, the EPOformulation of the invention includes a metal chelating agent such asEDTA or pentetic acid. The presence of a metal chelating agent in theEPO formulation can protect the protein from oxidation involving metalions and subsequent degradation.

According to still another embodiment of the invention, the EPOformulation of the invention includes an antioxidant. For example, thepresence of an antioxidant can reduce the extent of oxidation of the EPOformulation and reduce its degradation. Examples of antioxidants thatcan be used in embodiments of the invention include, ascorbate, sodiumbisulfite, sodium sulfite, butylated hydroxyltoluene (BHT), cysteine,methionine, thioglycerol or thioglycollic acid.

According to still another embodiment of the invention, the EPOformulation of the invention includes an antimicrobial preservative thatprevents microbial growth in the formulation during periods of prolongedstorage and handling.

The following examples are provided to illustrate the conjugates, andmethods and of the present invention, but not to limit the claimedinvention.

EXAMPLES Example 1

Preparation of Cysteine-PEG₂ (2)

1.1 Synthesis of Compound 1

Potassium hydroxide (84.2 mg, 1.5 mmol, as a powder) was added to asolution of L-cysteine (93.7 mg, 0.75 mmol) in anhydrous methanol (20 L)under argon. The mixture was stirred at room temperature for 30 min, andthen mPEG-O-tosylate of molecular mass 20 kilodalton (Ts; 1.0 g, 0.05mmol) was added in several portions over 2 h. The mixture was stirred atroom temperature for 5 days, and concentrated by rotary evaporation. Theresidue was diluted with water (30 mL), and stirred at room temperaturefor 2 h to destroy any excess 20 kilodalton mPEG-O-tosylate. Thesolution was then neutralized with acetic acid, the pH adjusted to pH5.0 and loaded onto a reversed phase chromatography (C-18 silica)column. The column was eluted with a gradient of methanol/water (theproduct elutes at about 70% methanol), product elution monitored byevaporative light scattering, and the appropriate fractions collectedand diluted with water (500 mL). This solution was chromatographed (ionexchange, XK 50 Q, BIG Beads, 300 mL, hydroxide form; gradient of waterto water/acetic acid-0.75N) and the pH of the appropriate fractionslowered to 6.0 with acetic acid. This solution was then captured on areversed phase column (C-18 silica) and eluted with a gradient ofmethanol/water as described above. The product fractions were pooled,concentrated, redissolved in water and freeze-dried to afford 453 mg(44%) of a white solid (1). Structural data for the compound were asfollows: ¹H-NMR (500 MHz; D₂O) δ 2.83 (t, 2H, O—C—CH ₂—S), 3.05 (q, 1H,S—CHH—CHN), 3.18 (q, 1H, (q, 1H, S—CHH—CHN), 3.38 (s, 3H, CH ₃O), 3.7(t, OCH ₂CH ₂O), 3.95 (q, 1H, CHN). The purity of the product wasconfirmed by SDS PAGE.

1.2 Synthesis of Compound 2 (Cysteine-PEG₂)

Triethylamine (˜0.5 mL) was added dropwise to a solution of compound 1(440 mg, 22 μmol) dissolved in anhydrous CH₂Cl₂ (30 mL) until thesolution was basic. A solution of 20 kilodalton mPEG-O-p-nitrophenylcarbonate (660 mg, 33 μmol) and N-hydroxysuccinimide (3.6 mg, 30.8 μmol)in CH₂Cl₂ (20 mL) was added in several portions over 1 hour at roomtemperature. The reaction mixture was stirred at room temperature for 24h. The solvent was then removed by rotary evaporation, the residue wasdissolved in water (100 mL), and the pH adjusted to 9.5 with 1.0 N NaOH.The basic solution was stirred at room temperature for 2 h and was thenneutralized with acetic acid to a pH 7.0. The solution was then loadedonto a reversed phase chromatography (C-18 silica) column. The columnwas eluted with a gradient of methanol/water (the product elutes atabout 70% methanol), product elution monitored by evaporative lightscattering, and the appropriate fractions collected and diluted withwater (500 mL). This solution was chromatographed (ion exchange, XK 50Q, BIG Beads, 300 mL, hydroxide form; gradient of water to water/aceticacid-0.75N) and the pH of the appropriate fractions lowered to 6.0 withacetic acid. This solution was then captured on a reversed phase column(C-18 silica) and eluted with a gradient of methanol/water as describedabove. The product fractions were pooled, concentrated, redissolved inwater and freeze-dried to afford 575 mg (70%) of a white solid (2).Structural data for the compound were as follows: ¹H-NMR (500 MHz; D₂O)δ 2.83 (t, 2H, O—C—CH ₂—S), 2.95 (t, 2H, O—C—CH ₂—S), 3.12 (q, 1H,S—CHH—CHN), 3.39 (s, 3H CH ₃O), 3.71 (t, OCH ₂CH ₂O). The purity of theproduct was confirmed by SDS PAGE.

Example 2

The following examples detail methods of modifying an EPO peptide thatis expressed in insect cells.

GnT1 and GalT1 Reaction in One Pot

2.1 Reaction in One Pot

The one pot GlcNAc transferase-1 and galactose transferase-1 reactionwas carried out by incubating insect-derived EPO (1 mg/mL) in 100 mMTris HCl pH 7.5 or MES pH 6.5 containing 150 mM NaCl, 5 mM UDP-GlcNAc, 5mM UDP-Gal, 5 mM MnCl₂, 0.02% sodium azide, 30 mU/mL of purified GlcNActransferase-1 and 200 mU/mL of purified galactose transferase-1 at 32°C. for 16 h.

2.2 Purification of EPO on Superdex 75

A Superdex 75 column was equilibrated in 100 mM MES buffer pH 6.5containing 150 mM NaCl at a flow rate of 5 mL/min. The EPO product fromstep 2.1 (above) was loaded on to the column and eluted with theequilibration buffer. The eluate was monitored for absorbance at 280 nmand conductivity. SDS-PAGE was used to determine which pooled peakfractions contains the EPO and used in further experiments.

2.3 ST3Gal-III Reaction

The ST3GalIII reaction was carried out by incubating 1 mg/mL EPO-Gal(from step 2.2, above) in 100 mM Tris HCl pH 7.5 or MES pH 6.5containing 150 mM NaCl, 0.5 mM CMP-N-acetyl-neuraminic acid-20kilodalton-PEG, 0.02% sodium azide, and 200 mU/mL of purified ST3Gal-IIIat 32° C. for 16 h.

Example 3

GnT1, GalT1 and ST3Gal-III (using CMP-NAN-20 KPEG) Reaction in One Pot

EPO (1 mg/mL), expressed in insect cells, was incubated with 30 mU/mL ofGlcNAc transferase-1, 200 mU/mL of galactose transferase-1 and 500 mU/mLof ST3GalIII with sugar nucleotides and CMP-N-acetyl-neuraminic acid-20Kd PEG in 100 mM MES buffer pH 6.5 and analyzed using SDS-PAGE. Similarto the results obtained in the two-step enzyme remodeling reactions,three bands of PEGylated EPO are seen in the one-pot, three enzymepreparations.

Example 4

Production of Biantennary PEG-EPO

4.1 Addition of GlcNAc to rEPO

Recombinant EPO, expressed in insect cells (1 mg/mL) in 0.1 M Tris, 0.15M NaCl, 5 mM MnCl₂ and 0.02% sodium azide at pH 7.2 was incubated with 3mM USP-GlcNAc, 50 mU/mg GlcNAc transferase-1 and 50 mU/mg GlcNActransferase-II at 32° C. for 24 h.

4.2 Addition of Galactose

To the GlcNAc-labeled peptide of step 8.1 (above) was added 3 mM UDP-Galand 0.2 U/mg galactose transferase-1. The mixture was incubated for 36 hat 32° C. The galactosylated product was isolated by gel filtrationchromatography on a Superdex 75 column in Tris-buffered saline. Thepurified product was concentrated to 1 mg/mL.

4.3 Addition of Sialic Acid or Sialic Acid PEG

The galactosylated product from step 4.2 (above) (1 mg/mL) in 0.1 MTris, 0.1M NaCl at pH 7.2 was incubated at 32° C. for 24 h with 200mU/mg ST3GalIII and 0.5 mM CMP-sialic acid or CMP-sialic acid-PEG (wherethe PEG has a molecular mass of 5 kDa, 10 kDa, 20 kDa or 30 kDa).

Example 5

N-Linked 30K PEGylation by CST-II

To EPO glycosylated as expressed in CHO (Chinese Hamster Ovary) cells (5mg, 0.166 μmol, 5 mL) was concentrated and buffer exchanged with trisbuffer (50 mM Tris, 0.15M NaCl, 0.001 M CaCl₂+0.005% NaN₃) to a finalvolume of 5 mL. Then CMP-sialic acid-PEG (30 kilodaltons, 25 mg, 0.833μmol; see FIG. 3B for structure of 30 Kdalton CMP-sialic acid-PEG), 0.25mL, 100 mM MnCl₂, 0.25 mL, and a bifunctional sialyltransferase fromCampylobacter jejuni, CST-II (1.4 U/mL, 0.5 mL, 0.7 U), were added. Theresulting mixture was rocked at 32° C. for 48 h.

At the conclusion of the reaction, the mixture was concentrated byultrafitration to 1 mL final volume, and was then buffer exchanged with25 mM NaOAc+0.005% Tween-80 (pH 6.0) to 2.5 mL. Q-Sepharose IEXchromatography was performed using 25 mM NaOAc+2M NaCl+0.005% Tween-80(pH 6.0) as eluent. Peak 2 was collected and concentrated to 1.5 mL byultrafiltration, then subjected to superdex-200 purification (column:Superdex 200, 16/60 GL, Amersham) using 1×PBS (pH 5.5+0.005% Tween80) aseluent. Peak 2 was collected and concentrated to 1.5 mL. This resultingmaterial was sterile filtered and formulated to a final volume of 2.5 mLusing 10 mM NaOAc (0.75% NaCl, pH 5.5). Protein concentration 264 μg/mL;660 μg protein was obtained (BCA determination).

Example 6

The following example illustrates a method for preparing O-linked 40kilodalton PEG linked EPO using ST3GalIII.

6.1 Desialylation

In this step EPO grown in Chinese Hamster Ovary cells (CHO cells), wasdesialylated. The GalNAc-Gal linkage serves as an acceptor for transferof the modified sialic acid PEG in step 6.2, below.

EPO solution 10 mL (10 mg, 0.33 μmol) glycosylated as expressed in CHO(Chinese Hamster Ovary) cells, was buffer exchanged with Tris buffer (20mM Tris, 50 mM NaCl, 5 mM CaCl₂, 0.02% NaN₃, pH 7.2) to give a finalvolume of 10 mL. Then 750 mU 2,3,6,8-neuramidase, from ArthrobacterUreafaciens, was added to the solution. The resulting mixture was rockedat 32° C. for 48 h. The product of this step was used directly in thenext step of the protocol (see below).

6.2 O-Linked 40K PEGylation

In this step ST3Gal2 is used to transfer a modified sialic acid-PEGmoiety to the desialylated EPO from step 6.1, above.

CMP-sialic acid-PEG (40 kilodalton, 33 mg, 0.825 μmol; see FIG. 3A forthe structure of 40 kilodalton CMP-SA-PEG), an O-glycan specificsialyltransferase (1.4 U/mL, 300 mU) (ST3GalI or ST3GalII), and 0.25 mLof 100 mM MnCl₂ were added to half of the above mixture. This mixturewas rocked at 32° C. for 48 h. After the 48 hour period, the reactionmixture was concentrated by ultrafiltration (MWCO 5K) to 2.8 mL, thenbuffer exchanged with 25 mM NaOAc+0.001% Tween-80, pH 6.0) to a finalvolume of 3 mL. The final product was ion exchange purified on SP (5 mL)three times (three injections, 1 mL each). PEGylated EPO (Peak 2) wascollected and concentrated by ultrafiltration to a final volume of 2 mLfor SEC purification. Purification on superdex 200 provided resolutionof the desired protein: EPO-GlcNAc-Gal-SA-PEG (40K) for the final stepof the reaction.

6.3 Terminal Sialylation of CHO-EPO-GalNAc-Gal-SA-PEG(40K)

In this step of the process sialic acid was added to the termini ofglycosyl structures not bearing a modified sialic acid residue.

Combined PEGylated EPO (approximately 2 mg from the reaction in step, babove) was concentrated by ultrafiltration (MWCO 5K) and then bufferexchanged with tris buffer (0.05M Tris, 0.15 M NaCl, 0.001 MCaCl₂+0.005% NaN₃) to a final volume of 2 mL. Then CMP-N-acetylneuraminic acid (CMP-NANA; 1.5 mg, 2.4 μmol), ST3GalIII (8.9 U/mL, 10μl, 0.089 U) and 50 μl of 100 mM MnCl₂ were added. The resulting mixturewas rocked at 32° C. for 24 h, then concentrated to 1 mL final volume.This solution was directly subjected to Superdex 200 purification using1×PBS (pH 5.5+0.005% Tween 80) as eluent. Peak 1 was collected anddiluted to 10 mL. Protein concentration was 52.8 ug/mL (BCA). A total of528 μg protein was obtained.

Example 7

In this example the pharmacokinetic profiles ofintravenously-administered CHO-derived EPO and glycoPEGylated variantsof the CHO-derived EPO were compared using an ELISA assay.

The pharmacokinetics of two non-PEGylated batches of CHO-derived EPO, a30K PEGylated CHO-derived erythropoietin produced by methods of theinvention, and 40K PEGylated CHO-derived Erythropoietin produced bymethods of the invention, were compared by ELISA after a single 30 μg/kgintravenous dose into rats. The measurement followed accepted ELISAprocedures using Europium detection.

7.1 Results

The Europium counts from the standard proteins from each plate were usedto generate a standard linear regression curve and equation. Theequation was used to convert the Europium count into the equivalent EPOquantity for each sample well.

The results are shown in FIG. 6. The limit of detection is approximately0.4 ng/mL for non-PEGylated EPO, and approximately 0.8 ng/mL for both 30kilodalton and 40 kilodalton PEGylated EPO.

Example 8

In this example the pharmacokinetic profiles ofsubcutaneously-administered CHO-derived erythropoietin (EPO), ahyperglycosylated non-glycoPEGylated EPO, an insect cell grownglycoPEGylated EPO, and a CHO cell derived glycoPEGylated EPO weredetermined using an ELISA assay.

Pharmacokinetics of a non-glycoPEGylated CHO-derived EPO, anon-PEGylated hyperglycosylated CHO derived EPO, a glycoPEGylated insectcell derived EPO; a 10K N-linked PEGylated insect cell-derivederythropoietin, and 40 kilodalton O-linked PEGylated CHO-derivederythropoietin were compared by ELISA after rats were given a single 10μg/kg subcutaneous dose.

8.1 Pharmacokinetic Results.

Results of these experiments are shown in FIG. 8, which shows theaverage quantity of EPO in ng/mL and the standard deviations in the ratserum samples at different time points after injection _(time=0 hour)for each EPO variant group. The limit of detection is approximately 0.3ng/mL for non-PEGylated EPO and PEGylated EPO.

Example 9

Purified 10K PEG-EPO and 20K PEG-EPO were spin-concentrated to 1 mg/mlusing Amicon-ultra-4 ultrafiltration tubes at 3,000 g at 4° C. They werethen diluted ten-fold into 20 mM universal buffer (4 mM glycine, 4 mMcitric acid, 4 mM HEPES (N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid)], 4 mM MES (2-[N-morpholine]ethane sulfonic)monohydrate)and 4 mM Tris hydrochloride (Tris [hydroxymethyl]-amino methane HCl)(fisher, lot # 040665) adjusted to between pH 4 and pH 8) to obtain 0.1mg PEG-EPO with having a pH range from pH 4 to pH 8 with 0.5 unit apart.

The solution was then filtered through a 0.22 μm syringe filter(Millex-GV) and 500 μl aliquots were filled in 2 ml HPLC vials. ThePEG-EPO samples with different pH were then put in 5° C. and 40° C.incubators. At the 2-week and 4-week time points, the stability ofPEG-EPO was analyzed by UV scan, SDS-PAGE, RP-HPLC (reverse-phase HPLC),and SEC-HPLC (size exclusion chromatography HPLC).

The absorption spectrum was obtained by an Amersham Ultraspec 4300 prospectroscopy using a 10 mm cuvette and a scanning rate of 4626 nm/min.Concentration of PEG-EPO was determined by Absorption at UV₂₈₀ using thefollowing equation:${{Conc}.\left( {{mg}/{ml}} \right)} = \frac{A_{280} - A_{340}}{1.2}$

The results of the UV scan experiments showed that there was nosignificant protein loss following 2 weeks of incubation at 5° C. and40° C.

Example 10

10K and 20K EPO-PEG samples were incubated at 40° C. for 0, 2 and 4weeks and the incubated samples were analyzed by SDS-PAGE. In theSDS-PAGE experiments, 4-20% Tris-glycine polyacrylamide gels fromInvitrogen were used. Briefly, 10 μL of 4× loading buffer was mixed with30 μl of sample and heated at 60° C. for 5 min. The mixture was thencooled down to room temperature and spun down by brief centrifugation,following which 30 μl of the mixture was loaded to the gel. For reducedSDS-PAGE, 100 mM DTT was added to the loading buffer. Theelectrophoresis was run for 1 hr at 200 V and the gels were stained withSafeblue coomassie staining solution. It was followed by iodine stainingby soaking the gel in 5% BaCl₂ solution for 5 min followed by additionof 4 ml 1/10 N iodine solution. Silver staining was then performed usingWAKO II silver stain kits.

Before incubation, there were no observable degraded bands in SDS-PAGEgels with either coomassie, iodine or silver staining prior toincubation. After 2 weeks at 40° C., a clear pH-dependent degradationpattern emerged for both 10K and 20K PEG-EPO samples. At lower pH (4 and5), bands with apparent MW between 64 K and 36 K were observed. The bandwith apparent MW of around 45 K was especially significant in the 10 KPEG-EPO samples. Iodine staining showed the existence of PEG moleculesin those bands. A band with apparent MW of 20 K in the 10 K PEG-EPOsample and one with apparent MW of 40 K in the 20 K PEG-EPO sample wereobserved in iodine staining gels. The fact that they were not in thecoomassie or silver stained gels suggested the existence of free sialicacid-PEG in the pH 4 samples incubated at 40 C. A different degradationpathway was found for samples at higher pH (≧7). For 10 K samples at pH7, 7.5 and 8, aggregate bands were apparent. The aggregate bandsdisappeared in reduced gel, indicating the disulfide-bond nature ofthese aggregate bands. No significant aggregate bands were observed forany of the 20 K samples. The same trend was observed for the 4-weeksamples as for 2-week samples. In summary, the SDS-PAGE resultsindicated that at pH equal or below 5, hydrolysis occurred in both 10 Kand 20 K samples, and at pH equal or above 7, aggregation occurred in 10K samples. 20 K PEG-EPO was more stable than 10 K PEG-EPO with respectto aggregation.

Example 11

RP-HPLC was run on a Zorbax C-3 column. SEC-HPLC was run on a TSKG5000PWxl column using 100 mM sodium acetate/150 mM NaCl at pH 5.5 asthe mobile phase with a flow rate of 0.5 ml/min.

In the RP-HPLC experiments, all 10 K and 20 K samples at various pHshowed the same PEGylation profile at 4° C. However, significantdegradation occurred at lower pH samples (pH 4, 5) when incubated at 40°C. A portion of Tri-PEG molecules, i.e., N-linked PEGylated isoformswhere the PEG molecules are attached to three branches of the glycosylresidues, were converted to di-PEG/mono-PEG or other species with ashorter retention time, which agreed with the observation of free(non-glycosylated) PEG by SDS-PAGE of low pH samples incubated at 40° C.

Because the SDS-PAGE indicated the instability of PEG-EPO when pH was ≦5or ≧7, emphasis was placed on comparing the results of pH 5.5, 6 and 6.5samples.

Aggregates were not detectable in 4° C. samples of both 10 K and 20 KPEG-EPO. Among pH 5.5, 6 and 6.5 samples, pH 6 and pH 6.5 samples hadless aggregates than pH 5.5 samples. The aggregate level in the pH 6.5sample was less than that in the pH 6 sample for 10 K PEG-EPO.

Example 12

The formulation of 10K PEG-EPO was studied further at pH 5.5, pH 6.0 and6.5 in acetate buffer (pH 5.5) and citrate buffer (pH 6.0 and 6.5) withNaCl and Tween 20. Several formulations of PEG-EPO, at varyingconcentrations and buffers were tested for their stability at severaltemperatures. The concentrations of the PEG-EPO samples tested are 0.2mg/ml and 0.5 mg/ml. The PEG-EPO samples were tested in one of threebuffers: 10 mM acetate/150 mM NaCl at pH 5.5 with 0.005% (w/v) Tween-20,10 mM citrate/136 mM NaCl, pH 6, 0.01% (w/v) Tween-20, and 10 mMcitrate/136 mM NaCl, pH 6.5, 0.01% (w/v) Tween-20. The formulations ofPEG-EPO (0.2 mg/ml and 0.5 mg/ml) were stored at 4° C., 30° C. for up to3 months and 40° C. for up to 5 weeks. The distribution of thetri+tetra-PEG EPO is shown below in Tables 1 and 2. TABLE 1 Tri- +tetra-PEG-EPO change in different formulations (0.2 mg/ml) (Startingpercentage of tri- + tetra-PEG-EPOis 89.6%) Tri- + tetra-PEG- Tri- +tetra-PEG- Tri- + tetra-PEG- EPO content after EPO content after EPOcontent after Formulation 5 weeks at 40° C. 3 months at 5° C. 3 monthsat 30° (0.2 mg/ml) (%) (%) C. (%) pH 5.5 83.5 (−6.8) 90.2 (0.7) 84.2(−6.0) acetate buffer pH 6 87.2 (−2.7) 89.8 (0.2) 88.8 (−0.9) citratebuffer pH 6.5 88.2 (−1.6) 90.1 (0.6) 91 (1.6) citrate buffer

TABLE 2 Tri- + tetra-PEG-EPO change in different formulations (0.5mg/ml) (Starting percentage of tri- + tetra-PEG-EPOis 89.7%) Tri- +tetra-PEG- Tri- + tetra-PEG- Tri- + tetra-PEG- EPO content after EPOcontent after EPO content after Formulation 5 weeks at 40° C. 3 monthsat 5° C. 3 months at 30° (0.2 mg/ml) (%) (%) C. (%) pH 5.5 79.7 (−11.1)90.0 (0.3) 84.1 (−6.2) acetate buffer pH 6 84.6 (−5.7) 90.0 (0.3) 88.6(−1.2) citrate buffer pH 6.5 84.5 (−5.8) 90.0 (0.3) 88.2 (−1.7) citratebufferThe numbers in parenthesis indicate the percent changes of tri- +tetra-PEG-EPO following incubation.

At T=5° C. and T=30° C., none of the formulations displayed anyaggregates or clips (degradation products) that were detectable bySDS-PAGE. At T=40° C., the pH 6.5 formulation displayed a higher amountof covalent aggregates than the pH 5.5 and pH 6 formulations, as well asa clip band detectable by SDS-PAGE.

The SEC-HPLC detected aggregates content in formulations were shown inTable 6 and 7. For 0.2 mg/ml formulation, analyzed by SEC-HPLC, therewere not aggregates deteceted at either 50° C. or 30° C. for 3 monthswhile 2.6%, 1.6%, 1.5% aggregates were measured for pH 5.5, pH 6 and pH6.5 formulation at 40° C. for 5 weeks, respectively.

For 0.5 mg/ml formulation, analyzed by SEC-HPLC, there were notaggregates deteceted at 5° C. while 22.2%, 9.8%, 8.1% aggregates weremeasured for pH 5.5, pH 6 and pH 6.5 formulation at 40° C. for 5 weeks,respectively. At 30° C. for 3 months, 4.6%, 1.8% and 0.9% aggregateswere detected for 0.5 mg/ml formulation. TABLE 3 Aggregate increase indifferent formulations (0.2 mg/ml) (0% aggregates at T = 0) Aggregateafter 5 Aggregate after 3 Aggregate after 3 Formulation weeks at 40° C.months at 5° C. months at 30° C. (0.2 mg/ml) (%) (%) (%) pH 5.5 2.6 0 0acetate buffer pH 6 1.6 0 0 citrate buffer pH 6.5 1.5 0 0 citrate buffer

TABLE 4 Aggregate increase in different formulations (0.5 mg/ml) (0%aggregates at T = 0) Aggregate after 5 Aggregate after 3 Aggregate after3 Formulation weeks at 40° C. months at 5° C. months at 30° C. (0.5mg/ml) (%) (%) (%) pH 5.5 22.2 0 4.6 acetate buffer pH 6 9.8 0 1.8citrate buffer pH 6.5 8.1 0 0.9 citrate buffer

Example 13

In order to test the stability of PEG-EPO formulations during freezethawing and agitation PEG-EPO in 10 mM acetate/150 mM NaCl at pH 5.5either with (0.4 and 0.2 mg/ml) or without 0.005% (w/v) Tween-20 (0.58mg/ml) were prepared. The freeze-thawing cycles were carried out byfreezing the samples at −20° C. and thawing at 4° C. three times. Theagitation experiments were carried out at either at 4° C. or 30° C. at200 rpm for 1 day, 2 days and 6 days.

The PEG-EPO formulations were stable over freeze-thawing cycles andagitation as judged by the SEC-HPLC and SDS-PAGE experiments. In thecase of the 0.58 mg/ml sample prepared without Tween-20, 6 days ofagitation resulted in a 0.4% increase in aggregate content. Nodegradation was observed in the other samples.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. An erythropoietin formulation comprising an erythropoietin peptide,wherein the erythropoietin peptide comprises a glycosyl linking groupattached to an amino acid residue of said peptide, said glycosyl linkinggroup comprising a modified sialyl residue having the formula:

wherein R² is H, CH₂OR⁷, COOR⁷, COO— or OR⁷ wherein R⁷ represents H,substituted or unsubstituted alkyl or substituted or unsubstitutedheteroalkyl; R³ and R⁴ are members independently selected from H,substituted or unsubstituted alkyl, OR⁸, NHC(O)R⁹ wherein R⁸ and R⁹ areindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl or sialic acid; L^(a) is alinker selected from a bond, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl; R¹⁶ and R¹⁷ are independentlyselected polymeric arms; X² and X⁴ are independently selected linkagefragments joining polymeric moieties R¹⁶ and R¹⁷ to C; X⁵ is anon-reactive group; and wherein the erythropoietin formulation shows nodetectable level of aggregation of the erythropoietin peptide asmeasured by SEC-HPLC after three months at a temperature of 5° C. orlower, or the erythropoietin formulation shows no substantial decreaseof tri- and tetra-PEG erythropoietin peptide as measured by RP-HPLCafter three months at a temperature of 5° C. or lower.
 2. Theformulation according to claim 1, wherein the moiety:

has a formula that is a member selected from:

wherein Q is selected from H and substituted or unsubstituted C₁-C₆alkyl; e and f are integers independently selected from 1 to 2500; and qis an integer from 0 to
 20. 3. The formulation according to claim 2,wherein said moiety has a formula that is a member selected from:

wherein Q is selected from H and substituted or unsubstituted C₁-C₆alkyl; e, f and f′ are integers independently selected from 1 to 2500;and q and q′ are integers independently selected from 1 to
 20. 4. Theformulation according to claim 1, wherein said glycosyl linker comprisesa glycosyl group having the formula:


5. The formulation according to claim 4, wherein said glycosyl group hasthe formula:

in which t is 0 or
 1. 6. The formulation according to claim 5, whereinsaid glycosyl linking group attached to said amino acid residue has theformula:

wherein AA is an amino acid residue of said peptide.
 7. The formulationaccording to claim 6, wherein said amino acid residue is a memberselected from serine or threonine.
 8. The formulation according to claim7, wherein said peptide has the amino acid sequence of SEQ. ID. NO:1. 9.The formulation according to claim 8, wherein said amino acid residue isa serine at position 126 of SEQ. ID. NO:1.
 10. The formulation accordingto claim 1, wherein said peptide comprises at least one said glycosyllinking group attached to an amino acid residue, said glycosyl linkinggroup comprising a glycosyl group having a formula selected from:

wherein R¹⁵ is said modified sialyl residue; and p is an integer from 1to
 10. 11. The formulation according to claim 10, wherein said at leastone glycosyl linking group attached to an amino acid residue of saidpeptide has a formula selected from:

and combinations thereof wherein AA is an amino acid residue of saidpeptide; t is an integer equal to 0 or 1; p is an integer from 1 to 10;and each R^(15′) is a member independently selected from H, OH, sialicacid, said modified sialyl residue and Sia-Sia^(p) wherein Sia^(p) issaid modified sialyl residue; wherein at least one R^(15′) is a memberselected from said modified sialyl residue and Sia-Sia^(p).
 12. Theformulation according to claim 11, wherein said amino acid residue is anasparagine residue.
 13. The formulation according to claim 12, whereinsaid peptide has the amino acid sequence of SEQ ID NO:1, and whereinsaid amino acid residue is an asparagine residue which is a memberselected from N24, N38, N83, and combinations thereof.
 14. Theformulation according to claim 13, wherein said glycosyl linking groupis attached to N24.
 15. The formulation according to claim 1, whereinsaid peptide is an erythropoietically active erythropoietin peptide. 16.The formulation according to claim 1, wherein said peptide isessentially non-erythropoietically active.
 17. The formulation accordingto claim 16, wherein said peptide is tissue protective
 18. Theformulation according to claim 1 further comprising a buffer.
 19. Theformulation according to claim 1, further comprising a buffer whereinthe buffer is selected from the group consisting of acetate, carbonate,citrate, glycinate, histidine, lactate, maleate, phosphate, succinate,tartrate, and tris.
 20. The formulation according to claim 1, whereinthe buffer is acetate, citrate, phosphate, or succinate.
 21. Theformulation according to claim 1, wherein the pH of the formulation isfrom about 5 to about
 7. 22. The formulation according to claim 1,wherein the pH of the formulation is from about 5.5 to about 6.5. 23.The formulation according to claim 1, wherein the buffer is acetate andthe pH of the formulation is about 5.5.
 24. The formulation according toclaim 1, wherein the buffer is citrate and the pH of the formulation isfrom about 6 to about 6.5.
 25. The formulation according to claim 1,wherein the buffer is from about 5 mM to about 100 mM.
 26. Theformulation according to claim 1, wherein the buffer is from about 5 mMto about 20 mM.
 27. The formulation according to claim 1 furthercomprising a tonicity adjusting agent that adjusts the tonicity of theformulation to be isotonic.
 28. The formulation according to claim 1further comprising a tonicity adjusting agent selected from the groupconsisting of sodium chloride, sodium sulfate, sucrose, trehalose,sorbitol, mannitol, histidine, arginine, and glycine.
 29. Theformulation according to claim 1 further comprising sodium chloride. 30.The formulation according to claim 1 further comprising a surfactant.31. The formulation according to claim 1 further comprising a surfactantselected from the group consisting of SDS, polysorbates and pluronics.32. The formulation according to claim 1 further comprising polysorbate20.
 33. The formulation according to claim 1 further comprising asurfactant in the range of about 0.001% to 1%.
 34. The formulationaccording to claim 1 further comprising a surfactant in the range ofabout 0.005% to about 0.01%.
 35. The formulation according to claim 1further comprising a stabilizer, metal chelating reagent, antioxidant,or an antimicrobial preservative.
 36. The formulation according to claim35, wherein the stabilizer is selected from the group consisting ofdisaccharide, amino acid, glycerol, albumin, and PEG.
 37. Theformulation according to claim 35, wherein the metal chelating reagentis EDTA or pentetic acid.
 38. The formulation according to claim 35,wherein the antioxidant is selected from the group consisting ofascorbate, sodium bisulfite, sodium sulfite, butylated hydroxyl toluene,cysteine, methionine, thioglycerol, and thioglycollic acid.
 39. Theformulation according to claim 1 comprising from about 0.1 mg/ml toabout 1 mg/ml said erythropoietin peptide, from about 5 mM to about 20mM sodium acetate or sodium citrate, from about 100 mM to 200 mM sodiumchloride, and from about 0.005% to about 0.01% polysorbate
 20. 40. Anerythropoietin formulation comprising an erythropoietin peptide, whereinthe erythropoietin peptide comprises a glycosyl linking group attachedto an amino acid residue of said peptide, said glycosyl linking groupcomprising a modified sialyl residue having the formula:

wherein R² is H, CH₂OR⁷, COOR⁷, COO— or OR⁷ wherein R⁷ represents H,substituted or unsubstituted alkyl or substituted or unsubstitutedheteroalkyl; R³ and R⁴ are members independently selected from H,substituted or unsubstituted 12 alkyl, OR⁸, NHC(O)R⁹ wherein R⁸ and R⁹are independently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl or sialic acid; s is an integerfrom 1 to 20; f is an integer from 1 to 2500; and Q is a member selectedfrom H and substituted or unsubstituted C₁-C₆ alkyl; and wherein theerythropoietin formulation shows no detectable level of aggregation ofthe erythropoietin peptide as measured by SEC-HPLC after three months ata temperature of 5° C. or lower, or the erythropoietin formulation showsno substantial decrease of tri- and tetra-PEG erythropoietin peptide asmeasured by RP-HPLC after three months at a temperature of 5° C. orlower.
 41. The formulation according to claim 40, wherein said modifiedsialyl residue has the formula:


42. The formulation according to claim 40, wherein Q is selected from Hand CH₃.
 43. The formulation according to claim 42, wherein saidglycosyl linking group has the formula:


44. The formulation according to claim 43, wherein s is 1; and f is aninteger from about 200 to about
 300. 45. The formulation according toclaim 40, wherein said amino acid residue is a member selected fromserine or threonine.
 46. The formulation according to claim 45, whereinsaid peptide has the amino acid sequence of SEQ. ID. NO:1.
 47. Theformulation according to claim 46, wherein said amino acid residue is aserine at position 126 of SEQ. ID. NO:1.
 48. The formulation accordingto claim 40, wherein said amino acid residue is an asparagine residue.49. The formulation according to claim 48, wherein said peptide has theamino acid sequence of SEQ ID NO:1, and wherein said amino acid residueis an asparagine residue which is a member selected from Asn 24, Asn 38,Asn 83, and combinations thereof.
 50. The formulation according to claim49, wherein said glycosyl linking group is attached to Asn
 24. 51. Theformulation according to claim 49, wherein each of Asn 24, Asn 38 andAsn 83 has said glycosyl linking group attached thereto.
 52. Theformulation according to claim 40 further comprising a buffer.
 53. Theformulation according to claim 40, further comprising a buffer whereinthe buffer is selected from the group consisting of acetate, carbonate,citrate, glycinate, histidine, lactate, maleate, phosphate, succinate,tartrate, and tris.
 54. The formulation according to claim 40, whereinthe buffer is acetate, citrate, phosphate, or succinate.
 55. Theformulation according to claim 40, wherein the pH of the formulation isfrom about 5 to about
 7. 56. The formulation according to claim 40,wherein the pH of the formulation is from about 5.5 to about 6.5. 57.The formulation according to claim 40, wherein the buffer is acetate andthe pH of the formulation is about 5.5.
 58. The formulation according toclaim 40, wherein the buffer is citrate and the pH of the formulation isfrom about 6 to about 6.5.
 59. The formulation according to claim 40,wherein the buffer is from about 5 mM to about 100 mM.
 60. Theformulation according to claim 40, wherein the buffer is from about 5 mMto about 20 mM.
 61. The formulation according to claim 40 furthercomprising a tonicity adjusting agent that adjusts the tonicity of theformulation to be isotonic.
 62. The formulation according to claim 40further comprising a tonicity adjusting agent selected from the groupconsisting of sodium chloride, sodium sulfate, sucrose, trehalose,sorbitol, mannitol, histidine, arginine, and glycine.
 63. Theformulation according to claim 40 further comprising sodium chloride.64. The formulation according to claim 40 further comprising asurfactant.
 65. The formulation according to claim 40 further comprisinga surfactant selected from the group consisting of SDS, polysorbates andpluronics.
 66. The formulation according to claim 40 further comprisingpolysorbate
 20. 67. The formulation according to claim 40 furthercomprising a surfactant in the range of about 0.001% to 1%.
 68. Theformulation according to claim 40 further comprising a surfactant in therange of about 0.005% to about 0.01%.
 69. The formulation according toclaim 40 further comprising a stabilizer, metal chelating reagent,antioxidant, or an antimicrobial preservative.
 70. The formulationaccording to claim 69, wherein the stabilizer is selected from the groupconsisting of disaccharide, amino acid, glycerol, albumin, and PEG. 71.The formulation according to claim 69, wherein the metal chelatingreagent is EDTA or pentetic acid.
 72. The formulation according to claim69, wherein the antioxidant is selected from the group consisting ofascorbate, sodium bisulfite, sodium sulfite, butylated hydroxyl toluene,cysteine, methionine, thioglycerol, and thioglycollic acid.
 73. Theformulation according to claim 40 comprising from about 0.1 mg/ml toabout 1 mg/ml said erythropoietin peptide, from about 5 mM to about 20mM sodium acetate or sodium citrate, from about 100 mM to 200 mM sodiumchloride, and from about 0.005% to about 0.01% polysorbate 20.